AN ABSTRACT OF THE DISSERTATION OF
Michael Dossett for the degree of Doctor of Philosophy in Horticulture presented on
April 22, 2011
Title: Evaluation of Genetic Diversity in Wild Populations of Black Raspberry
(Rubus occidentalis L.)
Abstract approved:
Chad E. Finn
In recent years, there has been renewed interest in black raspberry (Rubus
occidentalis L.) breeding. This has been spurred, in part, by an increase in black
raspberry consumption due to studies that have shown them to be particularly high in
anthocyanin content indicating high levels of antioxidants. Present cultivars are ill
adapted to the biotic and abiotic stresses of the Pacific Northwest, where the
commercial black raspberry industry is centered, and fields must be replanted after 35 seasons. Breeding progress in black raspberry has been limited by a lack of genetic
diversity. This work is the first documented effort to systematically collect and
evaluate wild R. occidentalis germplasm from across the native range of the species.
Sources of strong resistance to the virus vector Amphorophora agathonica Hottes
were identified from Maine, Michigan, and Ontario. Populations with outstanding
vigor and possible resistance to Verticillium wilt were identified, as were two
populations with a novel anthocyanin profile. In addition, variation was documented
for morphological traits such as thorniness and cane architecture as well as fruit
weight and gloss, and ripening times. Molecular analysis of genetic diversity using
microsatellite markers in these populations showed that black raspberry cultivars are
closely related to each other, but there is tremendous diversity in the wild
populations. The data also support phenotypic evidence that several “wild”
populations may be descended from feral cultivated plants. Overall, genetic diversity
in wild black raspberry germplasm shows tremendous potential for use in breeding
improved cultivars.
Evaluation of Genetic Diversity in Wild Populations of Black Raspberry (Rubus
occidentalis L.)
by
Michael Dossett
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented April 22, 2011
Commencement June 2011
Doctor of Philosophy dissertation of Michael Dossett
presented on April 22, 2011.
APPROVED:
Major Professor, representing Horticulture
Head of the Department of Horticulture
Dean of the Graduate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Michael Dossett, Author
ACKNOWLEDGMENTS
There are many people who have been instrumental in the completion of this
project. First, I would like to thank Dr. Chad Finn, for his tireless support and advice,
willingness to talk about small fruits at the drop of a hat, or just to listen, his constant
encouragement, and seemingly endless enthusiasm and optimism. I would also like
to thank Dr. Nahla Bassil for her guidance and support, enthusiasm, constant smile,
and cheerful disposition. I owe a great deal of what I have learned over the last
several years to them and appreciate all they have done to help me along the way. I
would also like to thank my other committee members, Dr. Shawn Mehlenbacher, Dr.
Rich Cronn and Dr. Ken Johnson, for their help in answering questions and
addressing problems that have arisen during the course of this project, and for their
insights that have helped point me in the right direction.
I also owe a debt of gratitude to Mary Peterson and Ted Mackey. Aside from
just plain putting up with me, their expertise in providing technical and field
assistance has been invaluable and is greatly appreciated. Andy Livesay, Rob
Livesay, Catherine Wright, Maeve Dempsey, Isaac Otto, and Alex Renn, all helped
me out too many times to count with watering plants when I was away, picking fruit,
and helping with field maintenance, for which I’m grateful. Erin Ortiz and Sumi
Maristany, in addition to providing valuable summer help, taught me a great deal
about how to manage my own time and supervise others, while being a joy to work
with.
I would also like to thank all the folks at the USDA-ARS Horticultural Crops
Research Lab and the USDA-ARS National Clonal Germplasm Repository,
particularly Bob Martin, Karen Keller, Jana Lee, Jerry Weiland, Paul Charron, Kim
Hummer, Joe Snead, Jeanine DeNoma, Missy Fix, Jim Oliphant, and Joseph
Postman. I would especially like to thank the past and present members of the Bassil
lab, April, Barb, Wambui, and Ted, with whom I have had the pleasure of working
over the last few years, for their help and camaraderie, as well as my fellow graduate
students in the Horticulture department at Oregon State University. Horticulture
department head Anita Azarenko, as well as Gina, LeeAnn, and Viki in the
Horticulture office have all been supportive and tremendously helpful with travel and
other paperwork.
I would like to thank Dr. Jungmin Lee and Chris Rennaker in Parma, Idaho,
who helped me with fruit chemistry analyses. In addition to letting me invade her lab
and doing her best to make me feel at home while working in Idaho, Jungmin has
been a great friend and colleague, given me advice whether I needed it or not, and
has always been there to support me in my research and career. Brian Yorgey, with
his good-natured humor, freezer space, and advice, also provided space and
equipment that helped a great deal during the course of this project.
I would also like to thank Dr. Chaim Kempler and Brian Harding with
Agriculture and Agri-Foods Canada, and Karen Keller with the USDA-ARS in
Corvallis, who helped me get started working with aphids, giving me advice, sharing
their techniques, and giving me tips on how to work with them.
Without the help of the following individuals sending me seed this project
would not have been possible: W. Anderson (Illinois), M. Bathrick (Pennsylvania), C.
Brown (New Jersey), P. Byers (Missouri), T. Cuff (Wisconsin), A. Dale (Ontario), R.
Davis (New York), R. Geneve (Kentucky), J. Hancock (Michigan), D. Handley
(Maine), A. Jamieson (New Brunswick), K. Kellogg (Connecticut), J. Lehman
(Indiana), T. Leslie (Ohio), H. Love (Tennessee), J. Luby (Minnesota), C. Mauchline
(Pennsylvania), R. Moyer (Virginia), G. Nonnecke (Iowa), M. Retter (Indiana), M.
Stanton (Ohio), H. Swartz (Maryland), F. Takeda (West Virginia), E. Thompson
(Arkansas), and C. Weber (New York). Financial support for this project was
provided by grants from the USDA-ARS Plant Exchange Office, USDA-ARS
Northwest Center for Small Fruits Research, and the Oregon Raspberry and
Blackberry Commission.
There are many others who have helped me and given their support and
encouragement these last years and I wish there was the space to list them all, so I
will finish by thanking my family, especially my wife Jaye, sisters Lisa and Michelle,
and mom and dad. Your support and love has meant more to me than you know and
was always there when I needed it the most; thank you.
CONTRIBUTION OF AUTHORS
Dr. Chad Finn oversaw and assisted with overall project conception, planning and
design. Dr. Jungmin Lee oversaw and assisted with sample preparation and analysis
of anthocyanins in black raspberry fruit (Chapter 4). Dr. Nahla Bassil oversaw and
assisted in the collection and analysis of microsatellite data (Chapter 5). Dr. Kim
Lewers provided primer sequences for EST-SSRs from an unpublished black
raspberry EST library for use in studying black raspberry diversity (Chapter 5). All
coauthors helped to edit and have approved the final version of the chapters with
which they are associated.
TABLE OF CONTENTS
Page
Chapter 1: Introduction ............................................................................................ 1
Chapter 2: Identification of Resistance to the Large Raspberry Aphid in Black
Raspberry................................................................................................................. 7
Abstract........................................................................................................................................ 8
Introduction.................................................................................................................................. 8
Materials and Methods ............................................................................................................... 11
Plant Materials and Aphids ..................................................................................................... 11
Aphid Resistance Screening.................................................................................................... 12
F1 Populations ........................................................................................................................ 14
BC1 Populations...................................................................................................................... 15
Statistics................................................................................................................................. 15
Results and Discussion ............................................................................................................... 15
References.................................................................................................................................. 21
Chapter 3: Morphology, Phenology, and Field Performance of Wild Black Raspberry
Germplasm ............................................................................................................ 30
Abstract...................................................................................................................................... 31
Introduction................................................................................................................................ 32
Materials and Methods ............................................................................................................... 35
Plant materials and field maintenance...................................................................................... 35
Collection of data and fruit samples ........................................................................................ 36
Statistics................................................................................................................................. 38
Results and Discussion ............................................................................................................... 38
Plant and fruit morphology...................................................................................................... 38
Flowering and fruiting phenology. .......................................................................................... 44
Vigor, disease resistance, and fruit weight. .............................................................................. 46
References.................................................................................................................................. 52
TABLE OF CONTENTS (Continued)
Page
Chapter 4: Characterization of a novel anthocyanin profile in wild black raspberry
mutants: an opportunity for studying the genetic control of pigment and color ....... 73
Abstract...................................................................................................................................... 74
Introduction................................................................................................................................ 74
Materials and methods................................................................................................................ 78
Plant materials and sample preparation.................................................................................... 78
Analysis of anthocyanins ........................................................................................................ 79
Results and discussion ................................................................................................................ 80
Conclusions................................................................................................................................ 88
References.................................................................................................................................. 88
Chapter 5: Genetic Diversity in Wild and Cultivated Black Raspberry Evaluated by
Simple Sequence Repeat Markers .......................................................................... 95
Abstract...................................................................................................................................... 96
Introduction................................................................................................................................ 96
Materials and Methods ............................................................................................................. 100
Plant Materials...................................................................................................................... 100
DNA extraction and amplification......................................................................................... 101
Data analysis ........................................................................................................................ 103
Results and Discussion ............................................................................................................. 103
Conclusions.............................................................................................................................. 112
References................................................................................................................................ 113
Chapter 6: Conclusions ........................................................................................ 131
Bibliography ............................................................................................................................ 136
LIST OF FIGURES
Figure
Page
3.1 Variation in fruit size, color, and gloss, in seedlings of wild Rubus
occidentalis L. populations grown in Corvallis, OR …………………………..70
3.2 Leaves from phenotypically normal Rubus occidentalis L. leaves, with
sessile leaflets (left), and leaves from a seedling of ORUS 3841, showing
leaflets attached to the main rachis by petiolules.……………………………...71
3.3 Flowering abnormalities segregating in ORUS 3801 ……………………….....72
4.1 Chromatograms showing anthocyanin profiles of black raspberry juice from
normal (ORUS 4141, rep 3), mixed (ORUS 4141, rep 4), and mutant
(ORUS 4143, rep 1; rr) samples from seedlings grown in Corvallis, OR,
USA and harvested in 2010..……………………………………………………93
4.2 Pathway for anthocyanin modifications in black raspberry …………………...94
5.1 Geographical distribution of 137 wild black raspberry (Rubus occidentalis
L.) populations surveyed……………...……………………………………...127
5.2 Unweighted pair-group method analysis (UPGMA) dendrogram depicting
all black raspberry (Rubus occidentalis L.) genotypes studied.………..…….128
5.3 Unrooted neighbor-joining dendrogram of black raspberry (Rubus
occidentalis L.) cultivars and closely paired wild accessions from a
condensed cluster in Fig. 5.2……………………………………….….……...129
5.4 Principal components plot of wild and cultivated black raspberry (Rubus
occidentalis L.) based on Euclidean distance measured from 21
polymorphic SSR loci and illustrating PCA clustering of wild accessions,
black raspberry cultivars, wild accessions clustered with cultivars in Fig.
5.3, and wild accessions clustering with ‘Explorer’ in Fig. 5.2…………..…..130
LIST OF TABLES
Table
Page
2.1 USDA-ARS plant introduction (PI) number, breeding program cross
identification (ORUS) number, general provenance, and results from
screening 132 wild black raspberry populations for resistance to the North
American large raspberry aphid………………………………………………...23
2.2 Summary of crosses in 2008 and 2009 involving black raspberry selections
resistant to the North American large raspberry aphid, showing breeding
program cross identification (ORUS) numbers and segregation for resistance...29
3.1 USDA-ARS plant introduction (PI) number, breeding program cross
identification (ORUS) number, general provenance, and year planted for 109
wild black raspberry (Rubus occidentalis L.) populations evaluated in
replicated field plots in Corvallis, OR…………………………………………..54
3.2 Vegetative and disease tolerance traits in 78 wild black raspberry (Rubus
occidentalis L.) populations and three cultivars grown in Corvallis, OR,
planted in 2007, and evaluated from 2008 to 2010……………………………..58
3.3 Vegetative and disease tolerance traits in 31 wild black raspberry (Rubus
occidentalis L.) populations and three cultivars grown in Corvallis, OR,
planted in the spring of 2008 and evaluated from 2008 to 2010.……………….62
3.4 Phenology and fruiting traits in 78 wild black raspberry (Rubus occidentalis
L.) populations and three cultivars grown in Corvallis, OR planted in 2007
and evaluated in 2009 and 2010.……………………………………………..…64
3.5 Phenology and fruiting traits in 31 wild black raspberry (Rubus occidentalis
L.) populations and three cultivars grown in Corvallis, OR, planted in 2008
and evaluated in 2010…………………………………………………………..68
3.6 Breeding program population identification (ORUS) number, state of origin,
and primocane-fruiting status of primocane-fruiting seedlings in wild black
raspberry (Rubus occidentalis L.) germplasm collected in 2006 and 2007 and
evaluated in the field in Corvallis, Oregon from 2008 to 2010…………………69
LIST OF TABLES (Continued)
Table
Page
4.1 Anthocyanin profiles and total anthocyanins by HPLC for juice from
seedlings of wild black raspberry populations from Lewis and Clark State
Park (ORUS 4141, Yankton, SD, USA) and Union Grove State Park (ORUS
4143, Beresford, SD, USA) and grown in Corvallis, OR, USA………………..92
5.1 U.S. Department of Agriculture - Agricultural Research Service plant
introduction (PI) number, accession name, origin, and type, for 137 wild
and 21 cultivated black raspberry (Rubus occidentalis L.) accessions studied..117
5.2 Summary information for 21 SSR primer pairs used for studying genetic
diversity in wild and cultivated black raspberry (Rubus occidentalis L.)
genotypes. ……………………………………………………………………..122
5.3 Allelic diversity, expected heterozygosity (He), observed heterozygosity (Ho)
and polymorphism information content (PIC) for 21 Rubus SSR primer pairs
in 21 cultivars and 125 wild black raspberry (Rubus occidentalis L.)
accessions……………………………………………………………………...124
5.4 Microsatellite alleles (fragment size in bp) at six loci in ‘Jewel’, ‘Haut’,
‘Allen’, ‘Earlysweet’, ‘Black Knight’, ‘Mac Black’ and related black
raspberry (Rubus occidentalis L.) cultivars, illustrating shared rare alleles
and discrepancies in reported pedigrees……………………….……………...126
Chapter 1:
Introduction
Michael Dossett
2
The black raspberry (Rubus occidentalis L.), often called “blackcap”, has
been cultivated in North America since at least the 1830s. The first record of its
domestication is the cultivar ‘Ohio Everbearer’, which was selected in the wild near
Lake Erie (Ohio) around 1832 for its unusual ability to produce an autumn crop. A
member of the Rosaceae, R. occidentalis is a diploid (2n=2x=14), and belongs to the
same subgenus (Idaeobatus) as the red raspberry (R. idaeus L.), with which it shares
the trait of its fruit separating cleanly from the receptacle. Black raspberries are
native to forest edges and disturbed habitats across eastern North America from the
Carolinas west to Arkansas and northeastern Oklahoma and north to Ontario and
New Brunswick (Jennings, 1988).
Black raspberry is a high value crop with a long history of production in the
US. In the early 1900s, production was centered in and around western New York,
with a large market for fresh and processed (dried) fruit. At that time, black
raspberry acreage in North America exceeded that of red raspberry. However by the
1940s, increasing disease problems led to a shift in production, with increased
acreage in Oregon and decreasing production in New York and the Ohio River
Valley. Since the 1960s, production has continued to decline across the US. This
decline has been largely attributed to disease and a lack of adapted, disease-resistant
cultivars. In Oregon, commercial black raspberry fields have an average planting life
of only 3-4 years (Halgren et al., 2007), half of what it was 60 years ago (Kuhlman
and Mumford, 1949). Two major disease problems have been attributed to the bulk
of decline in black raspberry production. Aphid-vectored viruses, particularly Black
3
raspberry necrosis virus (BRNV), are a leading reason for the short life of plantings.
At present, commercial fields in the Pacific Northwest become nearly 100% infected
with BRNV after just two seasons and subsequently experience serious decline
(Halgren, 2006). There are no cultivars with resistance to this virus or its vector, the
large raspberry aphid (Amphorophora agathonica Hottes). Wilt, caused by the fungi
Verticillium albo-atrum Reinke and Berth. and V. dahliae Kleb., is also a serious
problem in black raspberry production. Because these diseases cannot be easily
controlled through chemical or cultural practices, the best means for control is
through genetic resistance.
Public breeding of black raspberry began in 1893 (Jennings, 1988). At first,
many improved cultivars were developed, but breeding progress has slowed
dramatically in the last 60 years, and most of the old cultivars and selections have
since been lost. Since 1975, only four cultivars have been developed and released in
the U.S. The vast majority of acreage today is based on one cultivar, ‘Munger’,
developed and released in the 1890s.
Dossett (2007), reviewed black raspberry breeding and research, and with the
exception of a few studies further exploring the secondary metabolites in black
raspberry fruit and their potential health benefits, little has been published since. This
can probably be attributed to a decline in breeding efforts and research associated
with the decline in acreage. However, it may also be due to the frustration breeders
have expressed working with this crop in the past. Historically, progress in breeding
black raspberry has been limited by a lack of variation and segregation for important
4
traits in available elite germplasm. This has long been recognized and attempts to
broaden the genetic base of black raspberry date back to the 1950s. In contrast to red
raspberry, in which interspecific hybridization has played a major role in the
introduction of new traits of interest, this approach has been of limited success in
black raspberry. Slate and Klein (1952) tried crossing black raspberry to red
raspberry and found linkage drag to be a limitation. They concluded that when
selecting in backcross generations for types that resemble black raspberry, they were
unable to maintain selection for traits of interest. Working in Tennessee, Drain (1956)
crossed black raspberry with three Asian Rubus species. These hybrids had more
vigor and less winter injury than their black raspberry parents but tended to closely
resemble their Asian parents. Working in North Carolina, Williams (1950) also took
the approach of hybridizing with Asian species. While the resulting seedlings had
good vigor and improved disease resistance, they were almost completely sterile and
did not lead to any new cultivars. Ourecky and Slate (1966) took a slightly different
approach and crossed black raspberry with the closely related R. leucodermis Dougl.
ex Torrey & A. Gray, from western North America. This, too, proved to have its
limitations. They found that while the resulting F1 seedlings had very good vigor,
they also had nearly all the undesirable characteristics of R. leucodermis, including
dull, soft fruit of unremarkable size.
Ourecky (1975) considered a lack of genetic diversity in R. occidentalis to be
the biggest limitation for breeding in black raspberry, and despite the obstacles and
lack of success with interspecific hybridization for the improvement of black
5
raspberry germplasm, said that no future progress would be made in black raspberry
breeding without the use of germplasm from other species. With the recognition of
these limitations, it seems surprising that beyond a few selections made in the late
19th and early 20th Centuries, there is little record of the use of wild black raspberry
as a source of genetic diversity for breeding improved cultivars, or any effort to
systematically collect and evaluate wild black raspberry germplasm from across the
native range. Breeders have attempted small-scale efforts to collect and evaluate
regional black raspberry germplasm and this has been beneficial. Weber (2003) used
RAPD markers to show that some wild black raspberry selections from New York
were among the most genetically distinct of 16 black raspberry genotypes surveyed,
and Dossett et al. (2008) found significantly increased vigor and fewer disease
symptoms in progeny of a wild black raspberry selection from North Carolina when
compared to crosses among cultivars.
Surveying and characterizing wild black raspberry germplasm from across
the species’ native range is an obvious way of addressing the lack of genetic
diversity in black raspberry and is likely to yield results of more immediate benefit to
the industry than approaches relying on interspecific hybridization. In order for this
approach to be successful, however, a wide range of germplasm must be sampled
and intensively screened for traits of critical interest. In addition, the use of
molecular tools to gauge the level of diversity in this collection will be a critical part
of understanding how to best use the collection and where to focus further efforts.
With this in mind, we began to collect wild black raspberry germplasm from across
6
eastern North America in 2006. Evaluating this germplasm has been the focus of
work over the last four years and has yielded results which should have a lasting
impact on the black raspberry industry in Oregon and around the world.
7
Chapter 2:
Identification of Resistance to the Large Raspberry Aphid in Black
Raspberry
Michael Dossett and Chad E. Finn
Journal of the American Society for Horticultural Science
1018 Duke St.
Alexandria, VA 22314
135:438-444
8
Abstract
The large raspberry aphid, Amphorophora agathonica Hottes, is an important
vector of viruses in Rubus L. across North America. Although breeding for aphid
resistance has long been recognized as an important tool for protecting red
raspberries (Rubus idaeus L.) from viral infection, this is the first report of resistance
to A. agathonica in black raspberry (R. occidentalis L.). Seedlings from 132 wild
populations of black raspberries, representing the species’ native range, were
screened for resistance to A. agathonica. Strong resistance was found in three of
these populations, one from Ontario (ORUS 3778), one from Maine (ORUS 3817),
and one from Michigan (ORUS 4109). Resistance to the large raspberry aphid in
ORUS 3778 and ORUS 3817 is dominant and appears to be conferred by different
genes. We propose that the genes for resistance in ORUS 3778 and ORUS 3817 be
designated Ag4 and Ag5, respectively. Resistance to A. agathonica in ORUS 4109
also appears to be controlled by a dominant allele at a single locus, but cannot be
differentiated from Ag4 at this time.
Introduction
The black raspberry, often called “blackcap”, was first domesticated in the
1830s. A member of the Rosaceae, it is diploid (2n=2x=14) and belongs to the same
subgenus (Idaeobatus) as the red raspberry, with which it readily crosses. Native to
eastern North America from New Brunswick to the Carolinas and as far west as
9
Kansas and Nebraska, black raspberries typically are found in disturbed habitats and
near forest edges. The black raspberry industry in North America has undergone a
slow but steady contraction since the 1920s due in large part to disease and a lack of
adapted and resistant cultivars. Today, growers in Oregon, the leading production
region, typically see a decline in production after the second harvest and remove
fields after only three to four seasons because of decreased profitability (Halgren et
al., 2007). Much of this decline has been associated with aphid vectored viruses,
particularly black raspberry necrosis virus (BRNV) (Halgren et al., 2007), with
disease pressure so high that fields often become 100% infected within a couple of
growing seasons (Halgren, 2006).
Breeders have long recognized the importance of aphid resistance in the
development of new raspberry cultivars that protect against virus infection
(Schwartze and Huber, 1937). In Europe, at least 13 genes for resistance to the aphid
Amphorophora idaei Börner (Homoptera: Aphididae) are known (Hall et al., 2009),
and five biotypes of the aphid have been described (Hall et al., 2009, McMenemy et
al., 2009). Resistance to A. idaei is derived from a variety of sources, including red
and black raspberry. Keep (1989) indicated that the use of these resistance genes
have been very effective in slowing the spread of aphid vectored viruses in red
raspberry at East Malling, UK. At the Scottish Crop Research Institute (Invergowrie),
virus infection rates in plots of resistant cultivars was less than 10% after 3 years,
while 100% of susceptible plants became infected during the same period (Jones,
1976).
10
In North America, the primary vector of the raspberry mosaic virus complex
is the large raspberry aphid Amphorophora agathonica. Resistance to A. agathonica
is conferred by three genes, all from red raspberry. The single dominant gene Ag1 is
derived from the old UK cultivar Lloyd George (Daubeny, 1966). Genes Ag2 and Ag3
are dominant genes functioning in tandem to confer resistance and are derived from
wild R. strigosus Michx. populations in eastern Canada (Daubeny and Stary, 1982).
Sources of partial resistance to A. agathonica, as evidenced by the ability of aphids
to colonize plants in small numbers, have also been identified (Daubeny, 1972;
Kennedy et al., 1973) but the levels of resistance are less than that conferred by Ag1,
and so have received little attention. Until the 1990s, there was evidence for only a
single biotype of A. agathonica (Converse et al., 1971). Daubeny (1993), however,
described the discovery of an Ag1 resistance-breaking strain in British Columbia, and
new sources of resistance to A. agathonica are needed, not only to slow the spread of
resistant biotypes but to help maintain the durability of existing resistance as well.
While black raspberry has been an important source of resistance to A. idaei in
Europe (Keep and Knight, 1967), strong resistance to A. agathonica has not been
identified in R. occidentalis. Efforts to transfer resistance from red raspberry have
been slow and have not resulted in any cultivars. The purpose of this study was to
identify sources of strong resistance to A. agathonica in wild populations of black
raspberry and to determine its inheritance.
11
Materials and Methods
Plant Materials and Aphids. During the summer of 2006, friends and colleagues
living in eastern North America, within the native distribution of R. occidentalis,
were solicited to send seed or fruit from wild plants in their area. Additional seed
was obtained in 2007 through a similar request and collecting trips across the
southern and western edges of the native range (Hall et al., 2009). Through these
efforts, seeds were obtained from more than 150 locations across the range,
including 27 states and two Canadian provinces. From some locations, seed from
multiple maternal clones were sent as separate samples and kept separate from each
other after arrival. From other locations, the seeds represented bulk fruit samples of
many individuals (Table 2.1). Upon arrival, seeds were extracted from the fruit, dried,
and stored in a cool dry place until scarification. Seed was also obtained from eight
populations held at the National Clonal Germplasm Repository in Corvallis, OR.
Seeds were scarified in concentrated sulfuric acid for 45 min in an ice bath, rinsed
with sodium bicarbonate solution (Church and Dwight Co., Princeton, NJ, USA),
then soaked for 6 d in a 3 g·L-1 calcium hypochlorite solution (Sigma-Aldrich, St.
Louis, MO, USA) with an excess of calcium hydroxide (Mallinckrodt AR,
Phillipsburg, NJ, USA), then planted on moist peat and held at 4 oC for 8 weeks.
Seedling trays were then placed on the mist bench with bottom heat at 24 oC for
germination. After the emergence of their first true leaf, seedlings were planted in
72-cell trays (cell dimensions 4 x 4 x 6 cm), and then placed in the greenhouse under
16-h daylength. When seedlings were approximately 10-12 cm tall, they were
12
repotted individually and then subjected to screening for aphid resistance in the
greenhouse under similar conditions to those described above. Seedlings were
watered regularly and given a liquid fertilizer (20N–8.7P–16.6K; Scott’s, Marysville,
OH) once per week throughout this period to promote fast growth and tender tissue
attractive to aphids.
Alate and apterous A. agathonica were collected from a commercial red
raspberry field and were raised in screened cages on the susceptible ‘Meeker’ red
raspberry. Adult aphids from this colony were placed in petri dishes with a detached
leaf and moist filter paper to isolate nymphs as they were born. Nymphs were
removed from the petri dishes at 12-h intervals for 5 d and transferred to virus-free
plants of the susceptible black raspberry ‘Munger’ in a separate cage to ensure that
aphids used for screening seedlings did not carry viruses from the field. This colony
was maintained in a screened cage in the greenhouse under 16-h daylength for the
duration of the project. All aphids used in resistance screening came from this colony.
Aphid Resistance Screening. Between June and Aug. 2007, 5415 seedlings were
screened for resistance to A. agathonica in the greenhouse. An additional 1140 wild
seedlings were screened for resistance between Feb. and Apr. 2008. We began
screening 72 seedlings from each seed lot for resistance, but quickly determined that
seed lots could be adequately sampled for resistance with only 36 seedlings. Thus, in
27 seed lots, 72 seedlings were screened for resistance to A. agathonica, and in 151
seed lots, up to 36 seedlings were screened. Some seed lots experienced poor
13
germination; in cases where fewer than 36 seedlings were available, all seedlings
were screened for resistance. In those populations where seed from different
maternal clones at a location were sent as separate samples, 36 seedlings from each
were screened for resistance for a total of up to 216 seedlings from some locations.
The methods for screening plants for resistance to A. agathonica were similar to
those previously used by others screening for resistance to aphids in red raspberry
(Knight et al., 1959; Daubeny and Stary, 1982). Three apterous adult aphids were
placed on the youngest leaves of each seedling and allowed to remain undisturbed
for one week. After one week, each seedling was examined for evidence of
colonization. Plants with more than 10 aphids on them were scored as susceptible
and placed back on the bench. Plants with fewer than 10 aphids were marked and the
total number of adult and nymph aphids was recorded before plants were placed back
on the bench. Plants with fewer than three aphids had the appropriate number of
adult aphids added to them to bring the number up to three. Four days after this
initial inspection this procedure was repeated. Plants assumed to be susceptible
during the first inspection were re-examined to confirm susceptibility, while the
procedure for plants with fewer than 10 aphids remained the same. This was repeated
at 4-d intervals three more times for a total of 5 challenges. In this manner, plants
which continually exhibited a non-preference reaction by supporting fewer than 10
aphids were identified under conditions of extreme aphid feeding pressure.
In Apr. 2008, a colony of Amphorophora rubitoxica Knowlton was started
from aphids collected in the field, and maintained on ‘Munger’ using the same
14
procedures as outlined above. Single potted plants of ‘Munger’, ORUS 3778-1,
ORUS 3817-1, and ORUS 4109-1 were simultaneously placed in the colony and
monitored over a 2-week period for colonization. This was repeated three times over
the course of the summer to determine whether resistance to A. agathonica coincided
with resistance to A. rubitoxica. Seedlings from the cross ORUS 4158-2 × ORUS
3021-2 were also screened for resistance to A. rubitoxica using the same procedures
described above for A. agathonica.
F1 Populations. Two aphid resistant seedlings from ORUS 3778 (Simcoe, ON,
Canada) and ORUS 3817 (Gardiner, ME) were selected for crossing the following
year: ORUS 3778-1, ORUS 3778-2, ORUS 3817-1, and ORUS 3817-2 (a four digit
ORUS number followed by a dash and another number indicates a selection from
within that cross). These plants were moved to large pots and grown in the
greenhouse until September, when they were moved outside. In Jan. 2008, these four
plants were brought back into the greenhouse, along with potted plants of the
susceptible cultivars, Munger and Black Hawk. These plants were allowed to break
dormancy and flower, at which time crosses between the resistant and susceptible
genotypes were performed (Table 2.2). Fruit and seeds from these crosses were
treated as described above and, after two weeks drying, were subjected to the same
germination procedures. A subset of seedlings from each cross was screened in the
greenhouse for resistance to A. agathonica using the procedures described above
(Table 2.2). This procedure was repeated in Jan. 2009 with the single resistant
15
seedling from Bath, MI (ORUS 4109-1), and an aphid susceptible selection, ORUS
3021-1.
BC1 Populations. Five seedlings from each of four F1 populations (ORUS 4153,
ORUS 4155, ORUS 4157, and ORUS 4158) were moved to large pots in Sept. 2008.
The plants were fertilized weekly with a liquid fertilizer (20N–8.7P–16.6K; Scott’s,
Marysville, OH) and kept in a warm greenhouse under 16-hr daylength until 21 Nov.
2008 to promote growth and maturity. These seedlings were then treated to induce
flower bud initiation and dormancy before being brought back into the greenhouse
on 1 Apr. 2009 to begin flowering. In early May, crosses were performed between
these plants and field-grown plants of ORUS 3021-2 and ‘Jewel’ (Table 2.2). Seeds
and seedlings from these crosses were treated in the same manner as above and in
Dec. 2009, seedlings were screened for aphid resistance as already described.
Statistics. Chi-square tests to determine goodness-of-fit and probability of observed
segregation ratios were performed using SAS (version 9.1; SAS Institute, Cary, NC).
Results and Discussion
Greenhouse inoculation of black raspberry seedlings with A. agathonica and
A. rubitoxica resulted in colonization of susceptible plants. Susceptible plants
typically supported 20-30 aphids after the first week, and a few hundred after three
16
weeks. This was observed throughout the course of the screening periods and
allowed resistant genotypes to be readily identified.
Table 1.1 lists the populations screened, their origin, and the number of
seedlings found to have strong resistance to A. agathonica. Of the 132 populations
screened, strong resistance was confirmed in only three: ORUS 3778, ORUS 3817
and ORUS 4109. In addition, a single seedling from South Dakota, ORUS 4145-1,
was tentatively identified as resistant. Due to difficulties propagating this selection,
its resistance has not yet been confirmed and crosses involving ORUS 4145-1 to date
have been unsuccessful. Intermediate levels of colonization were noted on one or
more plants from several other locations, including ORUS 3827, ORUS 3844, ORUS
3848, ORUS 3854, ORUS 3855 and ORUS 4124. These may represent sources of
quantitative resistance, simply inherited weaker resistance, or may be an artifact of
changing environmental conditions and aphid populations in the greenhouse during
the three months in which screening took place. The 34 resistant seedlings of the 72
tested from ORUS 3778 averaged 1.9 aphids per plant after one week and 3.5 aphids
per plant per inspection over the course of the 23-day screening period (data not
shown). All 72 of the seedlings of ORUS 3817 were resistant. After one week, these
plants averaged 0.9 aphids per plant and averaged 2.1 aphids per plant per inspection
(data not shown) over the screening period. The single resistant seedling identified
from ORUS 4109 supported no aphids on each of the first four examinations, and
was found to have only two aphids on the final examination.
17
To confirm their resistance, three selections (ORUS 4109-1, ORUS 3778-1,
ORUS 3817-1) and ‘Munger’ (as a susceptible check) were propagated and five
plants of each were screened for aphid resistance in a completely randomized trial.
As expected, ‘Munger’ was the only plant to be colonized in this trial. Monitoring of
resistant seedlings of ORUS 3778 and ORUS 3817 in the field in Corvallis, OR
during Spring and Summer 2008 and 2009 failed to detect A. agathonica. While A.
agathonica numbers are fairly low in Corvallis, and vary greatly from year to year,
they were found on susceptible black raspberry seedlings in some adjacent plots in
2009, indicating their presence in the field.
Seedlings from the crosses of ORUS 3778-1, ORUS 3778-2, ORUS 3817-1
and ORUS 3817-2 with ‘Munger’ and ‘Black Hawk’ were all resistant to
colonization by A. agathonica when screened in the greenhouse (Table 2.2). This is
consistent with the hypothesis that resistance from these two sources is controlled by
dominant alleles and that all four of these resistant selections are homozygous.
Crossing these resistant progeny back to susceptible elite germplasm resulted in
segregation for resistance to A. agathonica. Both crosses involving resistance
originating from ORUS 3778 segregated 1:1 for aphid resistance in the BC1
generation (Table 2.2), confirming that resistance from this source is controlled by a
dominant allele at a single locus. The cross ORUS 3021-1 × ORUS 4109-1 also
segregated 1:1 (Table 2.2), confirming that resistance in ORUS 4109-1 is also
controlled by a single dominant gene for which ORUS 4109-1 is heterozygous. Due
to a lack of flowers on juvenile plants, only a single BC1 cross involving resistance
18
originating from ORUS 3817 was possible. Progeny of the cross ORUS 4158-2 ×
ORUS 3021-2 did not segregate 1:1 as expected. Instead, an excess of resistant
plants was observed. Nearly 64% of the progeny from this cross were resistant to
colonization by A. agathonica. Two possible explanations for this observation are
linkage to a lethal recessive allele originating in susceptible grandparents, or a gene
duplication event creating two loci acting independently to confer resistance in
ORUS 4158-2. Susceptibility to colonization by A. agathonica in this cross comes
from ‘Black Hawk’ and ‘Jewel’ as grandparents on opposite sides. If susceptibility
were linked to a lethal recessive allele shared by ‘Black Hawk’ and ‘Jewel’, this
would cause an excess of resistant plants as observed. This scenario seems plausible
given that black raspberry cultivars appear to be closely related (Dossett et al., 2010),
and that these two cultivars also share a rare recessive allele for non-glaucous canes
(Dossett, 2007). Alternatively, duplication of this locus could also cause an excess of
resistant progeny similar to that observed by Lunde et al. (2006) in ‘Zimmerman’
hazelnut for resistance to eastern filbert blight. If the duplicated loci are unlinked,
then 3:1 segregation for resistance should be observed. Linkage of the duplicated loci
would result in some lesser percentage of resistant progeny, depending on the
tightness of the linkage. Further study is needed to determine the exact reason for
excess resistant progeny in this cross. Observation of segregation ratios in progeny of
siblings of ORUS 4158-2 as well as in crosses involving resistant progeny of ORUS
4158-2 may clarify the cause of this result; however, it is clear from the data
presented that resistance to A. agathonica is dominant to susceptibility.
19
When single plants of the A. agathonica-resistant ORUS 3778-1 and ORUS
3817-1 and the susceptible ‘Munger’ were caged in a colony of A. rubitoxica,
‘Munger’ and ORUS 3778-1 became readily colonized while ORUS 3817-1 was
avoided entirely (data not shown). These results were echoed by observations from
an inadvertent infestation of this aphid species in the greenhouse in late Sept. and
Oct. 2008. Seedlings of ORUS 3778-1 and ORUS 3778-2 crossed with ‘Black
Hawk’ and ‘Munger’ became colonized by these aphids and the characteristic
chlorotic lesions from their feeding (Stace-Smith, 1954) were present on the leaves.
Seedlings of ORUS 3817-1 and ORUS 3817-2 crossed with ‘Black Hawk’ and
‘Munger’ remained free of aphids, and chlorotic lesions failed to appear. Testing in
Dec. 2009 showed that resistance to A. rubitoxica and A. agathonica cosegregated in
the cross ORUS 4158-2 × ORUS 3021-2. Amphorophora rubitoxica is not a known
virus vector and consequently is of little concern to growers in the Pacific
northwestern U.S. However, these results strongly suggest that resistance to A.
agathonica in ORUS 3778 and ORUS 3817 is controlled by separate genes,
(designated Ag4 and Ag5, respectively) with Ag5 from ORUS 3817 also conferring
resistance to A. rubitoxica. Resistance to A. agathonica in ORUS 4109-1 is not
effective against A. rubitoxica and cannot be distinguished from Ag4. A lack of
resistance found in the populations collected near this location suggests that
resistance in ORUS 4109-1 may be conferred by a unique gene, and further
investigation is necessary to determine if this is the case.
20
The occurrence of resistance to A. agathonica in R. occidentalis is of great
value to black raspberry breeders looking for resistance to BRNV and other aphid
vectored viruses in North America. While the data suggest the presence of minor
gene aphid resistance in some wild black raspberry populations, further study is
needed to determine its inheritance and the efficacy of using these sources
successfully. Because it only takes a very small number of aphids to colonize plants
and spread these viruses, partial resistance may have to be combined from many
populations to achieve the desired level of control, and even then may not be
sufficient. Work in Scotland has shown that even partial aphid resistance slows the
spread of aphid-transmitted viruses in red raspberry field plots (Jones, 1976, 1979).
Stace-Smith (1960), however, showed that partial resistance was not enough to
reduce virus transmission rates in greenhouse experiments with red raspberry.
The use of Ag4 and Ag5 together may increase their durability in new cultivars,
reducing the likelihood of new resistance-breaking aphid biotypes from appearing,
particularly if used in conjunction with minor gene resistance sources. This is an
important consideration. Birch et al. (2005) indicate that every type of major gene
aphid resistance in the UK has been broken while minor gene resistance has
remained durable, though less effective. Ag4 and Ag5 are also of potential value to
red raspberry breeders as they offer two new sources of resistance that may be
incorporated into breeding lines in much the same way as A10 (Keep and Knight,
1967). Screening wild black raspberry populations for additional sources of aphid
resistance is worthwhile. These data suggest that the range has not been exhaustively
21
sampled for diversity. Despite the resistance of all 72 plants sampled from Gardiner,
Maine, aphid resistance was not detected in three other nearby locations. To date,
every source of strong aphid resistance identified in R. occidentalis, and nearly all
those with some partial resistance, originate in the northern half of the species’ range
where raspberry aphids are typically more abundant and indicate this area may be a
good target for further exploration.
Small quantities of seed from most of the populations studied are available
from the USDA-ARS National Clonal Germplasm Repository in Corvallis, OR.
Plants of ORUS 3778-1 (PI 658505), ORUS 3817-1 (PI 658506), and ORUS 4109-1
(PI 659143) will also be available by request.
References
Birch, A.N.E., S.C. Gordon, R. Brennan, and A.T. Jones. 2005. Breeding for
resistance to the large raspberry aphid: An update on durability of current genes
and future prospects. International Organization for Biological and Integrated
Control/Western Palaearctic Regional Section Bul. 28(10):21-22.
Converse, R.H., H.A. Daubeny, R. Stace-Smith, L.M. Russell, E.J. Koch, and S.C.
Wiggans. 1970. Search for biological races in Amphorophora agathonica
Hottes on red raspberries. Can. J. Plant Sci. 51:81-85.
Daubeny, H.A. 1966. Inheritance of immunity in the red raspberry to the North
American strain of the aphid Amphorophora rubi Kltb. Proc. Amer. Soc. Hort.
Sci. 88:346-351.
Daubeny, H.A. 1972. Screening red raspberry cultivars and selections for immunity
to Amphorophora agathonica Hottes. HortScience 7:265-266.
Daubeny, H.A., and A.K. Anderson. 1993. Achievements and prospects – the British
Columbia red raspberry breeding program. Acta Hort. 352:285-293.
Daubeny, H.A. and D. Stary. 1982. Identification of resistance to Amphorophora
agathonica in the native North American red raspberry. J. Amer. Soc. Hort. Sci.
107:593-597.
22
Dossett, M. 2007. Variation and heritability of vegetative, reproductive and fruit
chemistry traits in black raspberry (Rubus occidentalis L.). MS thesis, Oregon
State Univ., Corvallis.
Dossett, M., N. Bassil, and C.E. Finn. 2010. Transferability of Rubus microsatellite
markers to black raspberry. Acta Hort. (In Press)
Halgren, A. 2006. Characterization, epidemiology, and ecology of a virus associated
with black raspberry decline. PhD diss., Oregon State Univ., Corvallis.
Halgren, A., I.E. Tzanetakis, and R.R. Martin. 2007. Identification, characterization,
and detection of black raspberry necrosis virus. Phytopathology 97:44-50.
Hall, H., K.E. Hummer, A. Jamieson, S. Jennings, and C. Weber. 2009. Raspberry
breeding and genetics. Plant Breeding Rev. 32:39-382.
Jones, A.T. 1976. The effect of resistance to Amphorophora rubi in raspberry (Rubus
idaeus) on the spread of aphid-borne viruses. Ann. Appl. Biol. 82:503-510.
Jones, A.T. 1979. Further studies on the effect of resistance to Amphorophora idaei
in raspberry (Rubus idaeus) on the spread of aphid-borne viruses. Ann. Appl.
Biol. 92:119-123.
Keep, E. 1989. Breeding red raspberry for resistance to diseases and pests. Plant
Breeding Rev. 6:245-321.
Keep, E. and R.L. Knight. 1967. A new gene from Rubus occidentalis L. for
resistance to strains 1, 2, and 3, of the Rubus aphid, Amphorophora rubi Kalt.
Euphytica 16:209-214.
Kennedy, G.C., G.A. Schaefers, and D.K. Ourecky. 1973. Resistance in red
raspberry to Amphorophora agathonica Hottes and Aphis rubicola Oestlund.
HortScience 8:311-313.
Knight, R.L., E. Keep, and J.B. Briggs. 1959. Genetics of resistance to
Amphorophora rubi (Kalt.) in the raspberry. I. The gene A1 from Baumforth A.
J. Genet. 56:261-280.
Lunde, C.F., S.A. Mehlenbacher, and D.C. Smith. 2006. Segregation for resistance to
eastern filbert blight in progeny of ‘Zimmerman’ hazelnut. J. Amer. Soc. Hort.
Sci. 131:731-737.
McMenemy, L.S., C. Mitchell, and S.N. Johnson. 2009. Biology of the European
large raspberry aphid (Amphorophora idaei): its role in virus transmission and
resistance breakdown in red raspberry. Agr. For. Entomol. 11:61-71.
Schwartze, C.D. and G.A. Huber. 1937. Aphis resistance in breeding mosaicescaping red raspberries. Science 86:158-159.
Stace-Smith, R. 1954. Chlorotic spotting of black raspberry induced by the feeding
of Amphorophora rubitoxica Knowlton. Can. Entomologist 86:232-235.
23
Table 2.1. USDA-ARS plant introduction (PI) number, breeding program cross identification (ORUS) number, general
provenance, and results from screening 132 wild black raspberry populations for resistance to the North American large
raspberry aphid.
Susceptible Resistant
PI no.(s)
ORUS no.(s)
Provenance
(no.)
(no.)
653296
4123
Mentone, AL
36
0
Not in GRINz
4184
Eureka Springs, AR
72
0
653327
3779
Litchfield County, CT
72
0
652975
4117
Clayton, GA
36
0
652976
4119
Clayton, GA
36
0
653294
4120
Clayton, GA
36
0
653298
4122
Dahlonega, GA
36
0
652977
4121
Union County, GA
36
0
653328
3780
Story County, IA
72
0
Not in GRIN
3789
Arenzeville, IL
6
0
653329
3781
Iroquois County, IL
70
0
553949
3946
Waukegan County, IL
4
0
653331
3796
Greene County, IN
72
0
653335
3800
Greene County, IN
36
0
653332
3797
Hendricks County, IN
36
0
653330
3794
Putnam County, IN
36
0
653333
3798
Sullivan County, IN
72
0
653334
3799
Vigo County, IN
36
0
653336
3801
southern IN
72
0
652984
4126
Alma, KS
6
0
653299
4124
Bonner Springs, KS
27
0
0
653303
4129
Fort Riley, KS
10
Table 2.1 (continued)
ORUS no.(s)
4127
4130
4128
4125
3802
3803
3804
3811
3812
3809
3808
3810
3806
3805
3807
3821
3820
3817
3819
3815
3814
3816
4109
4110
Provenance
Manhattan, KS
Minneapolis, KS
Ogden, KS
Perry Lake, KS
Fayette County, KY
Berkshire County, MA
Berkshire County, MA
Allegany County, MD
Anne Arundel County, MD
Dorchester County, MD
Harford County, MD
Howard County, MD
Howard County, MD
Washington County, MD
Washington County, MD
Camden, ME
East Vassalboro, ME
Gardiner, ME
Hallowell, ME
Monmouth, ME
Orono, ME
West Kennebunk, ME
Bath, MI
Benton Harbor, MI
Resistant
(no.)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
72
0
0
0
0
1
0
24
PI no.(s)
653301
651846
653302
653300
651848
653337
653338
653343
653344
Not in GRIN
653341
653342
Not in GRIN
653339
653340
653350
653349
653347
653348
651849
653345
653346
Not in GRIN
Not in GRIN
Susceptible
(no.)
2
16
3
3
72
72
36
36
36
4
72
36
36
36
72
72
72
0
36
36
36
72
35
36
Table 2.1 (continued)
PI no.(s)
553765
553766
Not in GRIN
553764
Not in GRIN
653323
653321
651847
653351
651851
653354
653355
653324
651850
653353
Not in GRIN
651852
653356
653357
653358
653359
653311
ORUS no.(s)
3948
3949
4111
3947
4112
4149
4148
4147
3823
3827
3828
3829
4150
3824
3826
3833
3830
3832
3835
3837
3838
4139
Provenance
Fred Russ State Forest, MI
Fred Russ State Forest, MI
Grand Ledge, MI
Oak Grove, MI
Okemos, MI
Belgrade, MN
Big Stone Lake National Wildlife Refuge, MN
Big Stone Lake State Park, MN
Cass County, MN
Dakota County, MN
Dakota County, MN
Dakota County, MN
Hasty, MN
Ramsey County, MN
Ramsey County, MN
Cassville, MO
Fordland, MO
Fordland, MO
Madison County, NC
Rutherford County, NC
Rutherford County, NC
Chadron, NE
Susceptible
(no.)
14
8
36
13
36
15
29
36
36
72
71
36
11
36
72
72
72
35
36
36
34
4
Resistant
(no.)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
25
Table 2.1 (continued)
ORUS no.(s)
4138
4136
4133
3952
4134
4137
4135
3955
3956
3843
3842
3839
3840
3841
3951
3844 – 3848x
3849
4107
4108
3854 –3855
3856
3851
3852
3853
Provenance
Chadron State Park, NE
Halsey, NE
North Loup State Recreation Area, NE
Otoe County, NE
Pibel Lake State Recreation Area, NE
Valentine, NE
Victoria Springs State Recreation Area, NE
Manasquan Reservoir, NJ
Tom's River, NJ
Columbia County, NY
Dutchess County, NY
Ontario County, NY
Ontario County, NY
Ontario County, NY
Poughkeepsie, NY
Yates County, NY
Clermont County, OH
Hilliard , OH
Newton Falls, OH
Centre County, PA
Centre County, PA
Chester County, PA
Greene County, PA
Somerset County, PA
Resistant
(no.)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
26
PI no.(s)
653310
653308
653305
606476
653306
653309
653307
638243
638244
653363
653362
653360
653361
Not in GRIN
618560
653364 – 653367y
653368
Not in GRIN
Not in GRIN
653372
653373
653369
653370
653371
Susceptible
(no.)
36
36
19
3
4
36
18
10
12
35
34
72
35
35
10
176
36
36
24
72
36
72
29
36
Table 2.1 (continued)
ORUS no.(s)
4185
4113
4114
4115 – 4116
4142
4145
4144
4146
4140
4143
4141
3904 – 3905
3914 – 3915
3857 – 3858
3859 – 3863
3864 – 3865
3866 – 3867
3868 – 3870
3871
3872 – 3876
3877 – 3882
3888 – 3891
3892 – 3895
3919
Provenance
Charlestown, RI
Glassy Mountain, SC
Glassy Mountain, SC
Rich Mountain, SC
Clay County State Park, SD
East Sioux Falls, SD
Newton Hills State Park, SD
Oakwood Lakes State Park, SD
Pease Creek State Recreation Area, SD
Union Grove State Park, SD
Yankton, SD
Cannon County, TN
Cheatham County, TN
Davidson County, TN
Davidson County, TN
Davidson County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
Grundy County, TN
Grundy County, TN
Henderson County, TN
Resistant
(no.)
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
27
PI no.(s)
Not in GRIN
652971
652973
652974, 653292
653315
653318
653317
653319
652988
653316
653314
653389
653395
653374
653375
653376
653377
653378
653379
653380
653381
653384
653385
653398
Susceptible
(no.)
5
36
36
72
14
2
36
8
11
13
29
72
72
72
180
72
108
108
72
175
216
144
140
36
Table 2.1 (continued)
Susceptible
(no.)
36
36
36
72
72
36
72
144
144
108
108
72
36
72
72
38
Resistant
(no.)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
34
PI no.(s)
ORUS no.(s)
Provenance
653396
3916
Unicoi County, TN
653397
3918
Unicoi County, TN
653382
3883
Van Buren County, TN
653390
3906 – 3907
Van Buren County, TN
653392
3909 – 3910
Van Buren County, TN
653393
3911
Van Buren County, TN
653394
3912 – 3913
Van Buren County, TN
653383
3884 – 3887
Warren County, TN
653386
3896 – 3899
Warren County, TN
653387 – 653388
3900 – 3903
Warren County, TN
653399
3926 – 3928
Columbia County, WI
653401
3930
Inwood, WV
653402
3931
Preston County, WV
653400
3929
Shepherdstown, WV
653325
3777
Mactaquac, NB, Canada
653326
3778
Simcoe, ON, Canada
y
- Multiple PI numbers indicate separate seed lots from this location in GRIN.
x
- Multiple ORUS numbers indicate seed lots from multiple maternal clones at this location were kept and evaluated
separately.
28
Table 2.2. Summary of crosses in 2008 and 2009 involving black raspberry selections resistant to the North American large
raspberry aphid, showing breeding program cross identification (ORUS) numbers and segregation for resistance.
z
Year
ORUS no.
2008
2008
2008
2008
2008
2008
2008
2009
2009
2009
2009
ORUS 4153
ORUS 4154
ORUS 4155
ORUS 4156
ORUS 4157
ORUS 4158
ORUS 4159
ORUS 4304
ORUS 4305
ORUS 4306
ORUS 4310
Parents
‘Black Hawk’
ORUS 3778-2
‘Munger’
‘Munger’
ORUS 3817-1
ORUS 3817-2
‘Black Hawk’
ORUS 4158-2
ORUS 3021-2
‘Jewel’
ORUS 3021-1
×
×
×
×
×
×
×
×
×
×
×
ORUS 3778-1z
‘Black Hawk’
ORUS 3778-2
ORUS 3817-1
‘Black Hawk’
‘Black Hawk’
ORUS 3817-2
ORUS 3021-2
ORUS 4153-1
ORUS 4153-1
ORUS 4109-1
Postulated
genotypes of
parents
ag4ag4 × Ag4Ag4
Ag4Ag4 × ag4ag4
ag4ag4 × Ag4Ag4
ag5ag5 × Ag5Ag5
Ag5Ag5 × ag5ag5
Ag5Ag5 × ag5ag5
ag5ag5 × Ag5Ag5
Ag5ag5 × ag5ag5
ag4ag4 × Ag4ag4
ag4ag4 × Ag4ag4
rr × Rr
Resistant
(no.)
Susceptible
(no.)
Expected
ratio
Chisquare
Probability
36
18
72
36
54
18
18
65
36
14
36
0
0
0
0
0
0
0
37
36
14
36
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:1
1:1
1:1
1:1
7.7
0.0
0.0
0.0
0.006
1.0
1.0
1.0
- A four digit ORUS number followed by a dash and another number indicates a selection from within that cross.
29
30
Chapter 3:
Morphology, Phenology, and Field Performance of Wild Black
Raspberry Germplasm
Michael Dossett and Chad E. Finn
31
Abstract
A lack of genetic diversity in cultivated black raspberry (Rubus occidentalis
L.) germplasm has been widely recognized as a major factor limiting breeding
progress towards developing improved cultivars. Despite this, little effort has been
made since the early 20th Century to systematically collect and evaluate wild black
raspberry for germplasm improvement. In recent years, there has been renewed
interest in black raspberry breeding to replace existing cultivars that lack durability
and disease resistance. In the fall of 2007 and spring of 2008, we planted seedlings
from 109 wild black raspberry populations, representing 24 U.S. states and two
Canadian provinces, in the field in a replicated planting in Corvallis, Oregon, to
evaluate performance. These populations showed wide variation in morphology,
plant architecture, fruiting season, vigor, and tolerance to Verticillium wilt. For
nearly every trait examined, wild black raspberry germplasm exhibited a range of
variation beyond existing cultivars, and showed great potential for use in future
breeding. While most populations were fairly uniform, segregation for fruit gloss and
possible tolerance to Verticillium wilt was noted in a few, indicating the possibility
of simple inheritance of these traits. Several populations with unusual morphology,
such as spinelessness or flower abnormalities, were identified, as were populations
that flowered on first year canes and produced fall fruit. Populations from the
southern edge of the range appear to be particularly well adapted to western Oregon,
with vigorous upright growth, strong canes, and many with a low incidence of
32
Verticillium wilt. This germplasm will be of great value to breeders interested in
developing improved black raspberry cultivars.
Introduction
Black raspberry has a long history as an important fresh and processed crop
in the U.S. In the early 1900s, black raspberry acreage in the U.S. exceeded that of
red raspberry (R. idaeus L.). However, in the years since, the black raspberry
industry has undergone a slow but steady contraction. This contraction has been
largely blamed on diseases and a lack of resistant cultivars to support the industry.
The first public breeding program for black raspberry improvement began in 1893
(Jennings, 1988). While many improved cultivars were developed in the early years
of this work, breeding progress has slowed dramatically in the last 60 years, and
most of the old cultivars and selections have been lost. Since 1975, only four new
cultivars have been developed and released, and the vast majority of acreage today is
based on a single cultivar, ‘Munger’, developed and released in the 1890s. This lack
of progress in breeding new black raspberry cultivars has been attributed to a lack of
variability in available elite germplasm and lack of disease resistance (Ourecky,
1975).
The limitations of elite black raspberry germplasm have been recognized for
many years, and attempts to broaden the genetic base of black raspberry date back to
the 1950s. In contrast to red raspberry, in which interspecific hybridization has
33
played a major role in the introduction of new traits, this approach has been of
limited success in black raspberry. Slate and Klein (1952) crossed red raspberry to
black raspberry and found linkage drag to be a major limitation, concluding that
when selecting for types that resemble black raspberry in backcross generations, they
were unable to also maintain traits of interest. Others crossed black raspberry with
Asian Rubus species (Drain, 1956; Williams, 1950). These hybrids had more vigor
and less winter injury than their R. occidentalis parents but tended to closely
resemble their Asian parents and were largely sterile. Ourecky and Slate (1966)
crossed R. occidentalis with the closely related R. leucodermis Dougl. ex Torr. &
Gray, from western North America. The resulting seedlings had good vigor, but also
had nearly all the undesirable characteristics of R. leucodermis, including dull, soft,
small fruit. More recently, Finn et al. (2003) surveyed R. leucodermis germplasm
from western Oregon and Washington, and selections from this work have been
valuable additions to the breeding program.
While it has been suggested that no further progress in black raspberry
breeding will be made without the use of germplasm from other Rubus species
(Ourecky, 1975), it has also been suggested that black raspberry germplasm has not
been sufficiently surveyed for the adaptation and disease resistance needed by the
industry (Daubeny, 1996). Surprisingly, beyond a few selections made in the late
19th and early 20th Centuries, there is little record of the use of wild black raspberry
as a source of genetic diversity for breeding improved cultivars. Despite the fact that
the species is widespread across the eastern half of North America (Jennings, 1988),
34
there is virtually no record of any effort to systematically collect and evaluate the
range of this germplasm since the first half of the 20th Century. Hedrick (1925) noted
193 black raspberry cultivars and selections, many of which were collected as wild
seedlings, and gave some information on their performance. However, in the
intervening time, the majority of this germplasm has been lost and fewer than two
dozen cultivars remain available today.
Some research has indicated the potential value of wild black raspberry
germplasm in developing improved cultivars. Dossett et al. (2008) found significant
improvements in vigor and disease resistance in progeny of a wild R. occidentalis
selection from North Carolina, NC 84-10-3. Dossett and Finn (2010) found strong
aphid resistance in wild black raspberry germplasm from Maine, Michigan, and
Ontario, showing the potential value of previously unexplored wild black raspberry
germplasm. Molecular studies have also indicated a degree of diversity in wild black
raspberry populations (Nybom and Schaal, 1990) and shown that cultivated black
raspberry may not be representative of the diversity found in wild germplasm
(Dossett et al., 2010; Weber 2003). The primary objective of this study was to
evaluate the performance of wild R. occidentalis germplasm collected from across
the species’ native range in North America and its potential for use in breeding
improved cultivars.
35
Materials and Methods
Plant materials and field maintenance. During the summer of 2006, friends and
colleagues living in eastern North America, within the native distribution of R.
occidentalis, (USDA NRCS, 2011) were solicited to send seed or fruit from wild
plants in their area. Additional seed was obtained in 2007 through a similar request
and from collecting trips across the southern and western edges of the native range
(Hall et al., 2009; Hummer et al., 2008a; 2008b). Through these efforts, seeds were
obtained from more than 150 locations across the range, including 27 states and two
Canadian provinces. From some locations, seeds from multiple maternal clones were
sent as separate samples and kept separate from each other after arrival. From other
locations, the seeds represented bulk fruit samples of many individuals (Table 3.1).
Upon arrival in the lab, seeds were extracted from the fruit, dried, and stored in a
cool dry place until scarification. Additional seed was obtained from all R.
occidentalis seed populations held at the National Clonal Germplasm Repository
(NCGR) in Corvallis, OR. Seeds were treated to promote germination as described
by Dossett and Finn (2010).
Seedlings were planted in the field in a randomized complete block design
with four replications representing 109 wild populations at the USDA-ARS North
Farm (Corvallis, OR). Plants were spaced 0.91 m apart in rows spaced 2.74 m apart.
Wild populations were represented by 3 - 4 plants per replication depending on the
number of available seedlings. Four plants each of the cultivars Jewel, Mac Black,
and Munger were included in each replication for comparison. Extra seedlings were
36
planted as border rows on either side of the field. Due to differences in timing of
seed acquisition, seedlings of 78 populations were planted in the field in September
2007, while 31 other populations were planted in the field in May 2008 (Table 3.1).
Seedlings planted in the field at different times were grown and maintained in two
separate, but adjacent, plots with independent randomization and replication. For
populations from which seed from multiple maternal clones was received separately,
seeds were germinated separately and seedlings were randomly chosen from each for
planting in the field. Plants were trained to a three-wire trellis system with a lower
wire at 0.50 m and two parallel wires hung 0.15 m apart at 0.91 m. Primocanes were
trained between the parallel wires and primocane tips were pruned approximately 510 cm above the wires to induce branching in early June, just before fruit ripening. In
the fall, primocane branches were pruned near the tips to help prevent them from
rooting in the row. Floricanes from the previous fruiting season were removed while
plants were dormant in the winter of 2009-2010. In late winter, new floricanes were
pruned so that floricane branches were approximately 30 cm in length. Plants were
fertilized, irrigated, and chemical weed controls applied per standard practices for
commercially grown black raspberries.
Collection of data and fruit samples. Evaluations of phenology and plant
performance were performed in a similar manner as Dossett et al. (2008). In 2009
and 2010, dates of first bloom (first fully open flower) and fruit ripening (first fully
colored fruit) were recorded for each plant. In addition, each plant was rated on a 1-9
37
scale for primocane vigor (1 = very poor vigor, 9 = extremely vigorous) in the spring
of both years. Symptoms of Verticillium wilt (Verticillium albo-atrum Reinke and
Berth. and V. dahliae Kleb) were recorded in early fall 2008, after the first season of
establishment, and again in the fall of 2009. Severity of symptoms was scored on a
0-6 scale (0 = no infection, 6 = all primocanes showing discoloration and stunting).
In the spring of 2009 and 2010, plants were scored on a 0-5 scale for the amount of
cane death over the winter (0 = no floricane death or injury to cane tips, 5 = 80%100% of floricanes dead). Samples of 25 randomly picked fruit were collected from
each plant for weighing and evaluation. Fruit were scored for gloss on a 1-5 scale (1
= most pubescent, 5 = most glossy). In a few cases, 25 ripe fruit were not available
from a given plant, so as many ripe fruit as could be collected were picked and
weighed. Fruit were picked when they were fully colored and separated readily from
the receptacle but before they were overripe. In 2009, plants were also rated on a 1-5
scale for primocane stiffness (1 = least stiff, 5 = most stiff) and lateral branching
angle (1 = ~75o-90o, 5 = < 15o). Other morphological features were recorded in the
field as they were noted. All traits were recorded in 2009 and 2010 for the seedlings
planted in 2007. Due to the immaturity of plants established in 2008, observations of
fruiting phenology, fruit characteristics, and winter floricane injury were recorded
for the seedlings during 2010 only. A single seedling from each of ORUS 3815,
ORUS 3827 and ORUS 4108 had morphological traits (e.g. canes with dense spines,
poorly set reddish/purplish fruit, differences in leaflet shape) indicating they were
probably the result of natural hybridization with wild red raspberry (R. strigosus
38
Michx.). Data from these individuals were excluded from analysis. A few off-types
of ‘Munger’ were noted, which ripened fruit approximately 7-10 days later than
expected for this cultivar and had a different flower cluster shape. Data from these
individuals were also excluded from analysis.
Statistics. Analysis of variance for all traits, and correlations between ratings of
Verticillium wilt symptoms, winter cane injury, vigor, branching angle, and cane
stiffness were performed using SAS (version 9.1; SAS Institute, Cary, NC).
Results and Discussion
There were significant differences among populations (P) for all traits in the
2007 and 2008 planted trials (Tables 3.2, 3.3, 3.4, and 3.5). For all the traits in both
trials that were evaluated in multiple years, there were significant differences due to
years (Y). While the population x year (P x Y) interaction was only significant for
vigor, and Verticillium wilt incidence in the 2007 and 2008 plantings and for winter
cane injury in the 2007 planting, results for both years are presented for all traits to
facilitate comparisons of traits that could only be evaluated for one year in the 2008
planting but could be evaluated for two years in the 2007 planting.
Plant and fruit morphology. Cane stiffness and branching angles are important
features for determining the overall growth habit of the plant. ‘Jewel’ and ‘Munger’
39
are fairly typical of most cultivars in having moderately stiff canes that branch at
~45o angles. While a direct statistical comparison between the 2007 and 2008
plantings is not possible, means for the standard cultivars were similar between fields
and some inferences can be made. There was wide variation for both of these traits in
the wild populations studied with a broader range in the 2008 planting (range 1.0-5.0
and 1.0-4.8 for stiffness and branching angle respectively, Tables 3.2 and 3.3). Plants
from the western edges of the range (Nebraska, South Dakota, and western
Minnesota) had a strong tendency toward having a more open sprawling plant habit
with a combination of wide branching angles and more limber canes. Plants from the
southern parts of the range had a tendency toward stiffer canes and narrower
branching angles, resulting in very erect plants. The 2008 planting was primarily
comprised of populations from the southern and western fringes of the range, which
accounts for the broader range of means over the 2007 planting, despite there being
fewer populations in this group. ORUS 3805 was noted for having exceptionally stiff
upright growth. These seedlings did not have the narrow branching angles often
associated with this characteristic but had stiff self-supporting canes to a greater
degree than any of the other populations studied. To date, this trait has not been
closely examined, probably because there is little variation in existing germplasm;
however, differences may affect suitability for machine harvest by holding fruit
further away from the crowns of the plants where they are less likely to fall between
the catch plates of the harvester. Stiff, strong canes may be desirable if it leads to less
cane breaking or damage from machine harvesters or other equipment. Having canes
40
at a steep vertical angle may create a dense canopy and may make plants more
susceptible to cane anthracnose (caused by [Elsinoe veneta {Burkholder} Jenk.]) or
other diseases. Cane stiffness, and cane branching angle were strongly correlated (r =
0.27, p<0.001) and cane stiffness was also correlated with vigor scores (r = 0.33,
p<0.001). This is not entirely surprising, as more vigorous plants have a tendency to
produce thicker, and therefore stiffer, canes.
Fruit morphology varied widely in the populations studied (Tables 3.4 and
3.5). The presence of fine hairs, or pubescence, on the surface of the drupelets is a
significant factor affecting how glossy the fruit appears. Hairs between the drupelets
interlock and are an important part of fruit cohesion in raspberries because the
receptacle is retained on the plant when fruit are picked (Jennings, 1988; Hall et al.,
2009). For the commercial processing industry, fruit glossiness is probably
unimportant because most of the fruit is processed into juice or puree. For the fresh
market, however, glossy fruit are more attractive to consumers. Several populations
had exceptionally glossy fruit (e.g. ORUS 3800, ORUS 3838, ORUS 3842, and
ORUS 4114) while others tended to have very pubescent fruit (e.g. ORUS 3930,
ORUS 4119, and 4115-16). While the populations studied tended to be uniform in
fruit gloss, several populations (e.g. ORUS 3778, ORUS 3781, ORUS 3797, and
ORUS 4123) segregated strongly for this trait (Fig. 3.1) indicating that there may be
one or a few loci accounting for much of the variation observed.
Anthocyanin deficient black raspberries with yellow or orange fruit have
been noted in the past and are generally regarded as of interest only because of their
41
novelty. Though studies have shown yellow fruit in R. occidentalis to be controlled
by the same locus as in red raspberry, commercial yellow raspberries are all derived
from anthocyanin deficient red raspberry. Nevertheless, it is worth noting that
segregants for yellow/orange fruit were identified in three of the populations studied
(ORUS 3780, ORUS 3839, and ORUS 3849).
In addition to the differences in plant morphology and structure that were
scored in all individuals, several populations were noted for differences in
morphology from what is typically observed in black raspberry. Three populations
(ORUS 3805, ORUS 3857-58, and ORUS 3877-82) were noted for having seedlings
with semi-smooth canes. While there was a range of spine densities within these
populations, a large percentage of seedlings from each of these populations had
either no or very few spines along the cane. These seedlings still had spines along the
basal sections of the canes (~30 cm and lower), and had a tendency to retain a few
small soft spines along the petioles. While spinelessness is not a major breeding
objective for the processing industry, black raspberries grown for processing are still
hand-tipped before harvest, and spineless canes would help increase the speed and
efficiency of handling canes. A long term goal of breeding programs is to develop
large fruited types that would be better suited to fresh market and individually quick
frozen (IQF) market. If fruit are to be harvested by machine for the IQF market, as
with blackberries, thornlessness becomes increasingly important in this litigious
society. Thornlessness would also be valuable for plants managed for fresh market
and you-pick operations where there is more non-mechanized handling of canes.
42
Spinelessness has received some interest historically. Card (1898) noted a few
cultivars for spinelessness, though all historic sources of this trait have been lost. In
red raspberry, spinelessness results from the recessive gene s in the old cultivar
‘Burnetholm’ (Lewis, 1939). This source has been used extensively for breeding
spineless red raspberries and there have been efforts to introduce this trait into black
raspberry (Jennings et al., 1986). To date, progress using this source of spinelessness
for breeding black raspberry has been slowed by linkage drag. The cultivar
‘Hortiberry1’ (‘EbonyTM’) was recently released in New Zealand using this source of
spinelessness, however, its fruit are purplish and very pubescent (Hall et al., 2009).
Canes of R. occidentalis typically have a waxy glaucous coating. Lewis
(1939) described gene B which confers the waxy bloom on the canes of some red
raspberries. Dossett (2007) noted non-glaucous canes from progeny of ‘Dundee’,
‘Hanover’, and ‘Jewel’ indicating that these cultivars are heterozygous for a gene
homologous to gene B. Seedlings from ORUS 3799, ORUS 3844-48, and ORUS
4124 were observed to segregate for non-glaucous canes. This same feature was
noted in a wild plant from Minneapolis, KS, but was not noted in seedlings
germinated from that population (ORUS 4130). Because of the rarity of this trait in R.
occidentalis, it seems likely that the presence of seedlings with non-glaucous canes
may be an indication that these populations could have some cultivated ancestry.
Jennings (1988) noted that this waxy bloom is associated with a low incidence of
certain cane diseases (presumably in red raspberry), but it is not clear whether this is
due to the physical presence of the wax or linkage to resistance genes.
43
In addition to differences in cane morphology, some variations in leaf
morphology were also noted. Two seedlings from Ontario County, NY (ORUS 3841)
were noted for having petiolules attached to the leaflets rather than being sessile to
the main petiole as is normally the case (Fig 3.2). This same feature was noted on a
single wild plant growing near Hasty, MN (Hummer et al., 2008b). This feature was
not observed in seedlings from any of the other populations, and may be due to
homozygosity for recessive alleles at an as of yet undescribed locus. Several
populations from eastern South Dakota and western Minnesota (ORUS 4141, ORUS
4143, ORUS 4144, ORUS 4147, ORUS 4148, and ORUS 4149) also had unique leaf
morphology, segregating for plants with curled leaf edges. The segregation observed
in these populations along with the regional occurrence suggests a genetic cause.
Approximately 30% of the seedlings from ORUS 3801 had unique flower
morphology. In the affected seedlings, sepals were large and leafy (Fig. 3.2), not
unlike what might be expected for gene L1 in red raspberry (Jennings, 1966).
However, other morphological features of these flowers do not match this
description. Flowers on the affected plants developed five elongated and reflexed
stamens in place of petals (Fig. 3.3) and had fewer pistils than on normal plants.
Fruit developing from these flowers lacked the fleshy drupelets of a normal black
raspberry fruit, and instead had just a thin but firm and only slightly juicy covering
over the seed. While this mutation has no apparent value for black raspberry
breeding, it may be of interest to biologists studying the genetic control of flower
development in angiosperms.
44
Flowering and fruiting phenology. One of the limiting factors for black raspberry as
a fresh market fruit is the short harvest season offered by present day cultivars.
Available cultivars all begin to ripen their fruit within an approximately 2-3 week
window in late June and early July. ‘Mac Black’ is unusual in that it begins to ripen
about two weeks later than most other cultivars. Because of this, it has found a niche
for extending the season for fresh fruit. In Oregon, where most fruit is processed,
growers would like a replacement for ‘Munger’ with a slightly earlier season to
provide a little more separation between the black raspberry and blackberry season
and to decrease the likelihood that black raspberry harvest will overlap with hot
weather. In very hot weather, black raspberry fruit tends to dry up onto the torus and
become difficult to harvest.
Flowering and ripening times were similar between fields for the three
cultivars examined in 2010, (Tables 3.4 and 3.5), while wild populations ranged
from being about a week earlier than ‘Munger’ (ORUS 3826, ORUS 3892-95,
ORUS 3929) to being nearly as late as ‘Mac Black’ (ORUS 3801). In 2010,
unusually warm temperatures leading up to flowering, followed by much colder
temperatures during bloom stretched the period out so that about five weeks elapsed
between the earliest flowering genotypes (ORUS 3826) and the latest (‘Mac Black’).
In contrast, in 2009, the date of first bloom occurred over approximately three weeks.
Black et al. (2008) developed a heat unit model for predicting bloom in Rubus and
noted that the R. occidentalis selections in their study ranged from being among
45
those genotypes that required the fewest growing degree hours, to among the most.
Using a similar model calculated on a daily rather than an hourly basis (due to
availability of data), only 230 growing degree days accumulated from 15 May - 15
June 2010 compared to 315 during the same period in 2009 (data not shown). This
difference in temperature and heat unit accumulation probably accounted for the
differences observed in the duration of bloom.
The fruiting season was also a few days later in 2010 than in 2009, again,
probably due to cooler than average temperatures from late May through most of
June (Tables 3.4 and 3.5 ). Black et al. (2008) were unable to extend their model to
fruit ripening, noting that light and environmental influences likely complicate the
relationship between temperature and fruit ripening. Despite the differences in
flowering and ripening dates among populations between 2009 and 2010, their rank
was similar and the population by year interactions for this trait were not significant
(p=0.08 and p=0.13, respectively). The lack of a population x year interaction was
similar to what Dossett et al. (2008) found in populations from a black raspberry
diallel. The lack of interaction and high heritability of these traits, indicates that
selecting for early or late fruiting should be fairly straightforward without the need
for multiple years of observation (Dossett et al., 2008). However, selecting for earlier
or later season may be made more difficult by exceptional weather conditions such
as those noted by Finn et al. (2003), where two weeks separated the earliest and
latest flowering R. leucodermis genotypes in one year while bloom occurred over a
three day period in another.
46
Primocane-fruiting, or the ability to bear fruit in the fall on first year canes, is
an unusual trait in black raspberry, but has been noted in the past (Dossett and Finn,
2011). While not well suited for the processed black raspberry industry, primocane
fruiting extends the season substantially and is valuable for fresh market raspberry
and blackberry producers. Dossett and Finn (2011) reviewed the occurrence of
primocane-fruiting in black raspberry and noted that this trait is absent in
commercially available cultivars but several wild populations were sources of this
trait. While the ripening dates for primocane fruit were not recorded, the presence or
absence of primocane fruit in the populations studied was noted (Table 3.6).
Vigor, disease resistance, and fruit weight. With a few exceptions, primocane vigor
was higher in 2009 than in 2010 for nearly every population (Tables 3.2 and 3.3).
While a few populations maintained good vigor across years (e.g. ORUS 3892-95,
and ORUS 4122), the cultivars, and most of the populations, dropped significantly
(Tables 3.2 and 3.3). Verticillium wilt infection was widespread in the field and was
probably the major reason for this decline, however, viruses or other factors may
have played a role. The strong population by year interaction also indicates that
successful selection for this criterion will likely require multiple years of observation,
or waiting until a field is 3-4 years old to see which genotypes have declined (Tables
3.2 and 3.3). Unfortunately, while it makes sense to wait a few years to evaluate a
seedling field when selecting for plant durability, this is not always economically or
logistically feasible in a breeding program. Populations with excellent vigor were
47
identified in both plantings, and tended to be more prevalent from Tennessee, South
Carolina, and Georgia, along the southern portions of the range. Dossett et al. (2008)
also noted excellent vigor in seedlings of a wild selection from North Carolina,
further supporting the idea that plants from this region may be particularly welladapted to the Willamette Valley.
Populations varied widely in their response to Verticillium wilt (Tables 3.2
and 3.3). The ratings in 2008, after the first season of establishment, were not at all
indicative of disease progression over the following two years. Several populations
(ORUS 3780, ORUS 3801, and ORUS 3817, to name a few) appear to be
particularly sensitive to Verticillium wilt, with high disease ratings and more than
50% of the seedlings dying from infection by the end of 2010. At the opposite end of
the spectrum, ORUS 4115-16, from Rich Mountain, SC, appeared to be resistant or
at least highly tolerant of the disease with excellent vigor, and almost no disease
symptoms recorded in 2008 or 2009 (Table 3.3). While there were several other
populations that appeared to hold up well to Verticillium wilt (Tables 3.2 and 3.3),
none was immune to the disease, and the three cultivars examined, ‘Munger’, ‘Jewel’,
and ‘Mac Black’, all appeared to fall somewhere in the middle - neither particularly
susceptible nor resistant.
In several populations (e.g. ORUS 3777, ORUS 3931, and ORUS 4133),
individual plants were severely affected by Verticillium wilt and showed many
disease symptoms while adjacent plants appeared healthy. Because seedlings were
not inoculated with the fungus prior to planting and were instead exposed to variable
48
levels of the fungus in the soil, these could represent escapes from infection, or they
could represent some sort of resistance. In the trailing blackberry, R. ursinus Cham.
& Schltdl., there is good evidence that resistance to Verticillium wilt is a simply
inherited trait (Wilhelm and Thomas, 1950; Wilhelm et al., 1965). On the other hand,
Fiola and Swartz (1994) found evidence that resistance in red raspberry was
quantitatively inherited. Understanding sources of resistance to Verticillium wilt and
its inheritance in R. occidentalis are areas needing further study and should be a
priority for the industry. The development of a quick and affordable screening
procedure for black raspberry seedlings would greatly facilitate this work, as current
methods are not feasible for studying resistance to Verticillium wilt on a large scale.
In Oregon, one of the biggest disease symptoms noted by growers is the failure of
floricanes to leaf out in the spring. This has been attributed at various times to cold
and to disease and likely has many causes. The degree of winter floricane injury in
this study varied widely with some populations (e.g. ORUS 4115-16, Table 3.3)
showing almost no damage to the floricanes while others were severely damaged (e.g.
ORUS 3780, ORUS 3817, and ORUS 3124, Table 3.2). Whatever the cause, it
appears that there is considerable room for improvement over the cultivars, which all
suffered considerable cane death (rated 1.7-2.4), particularly in Spring 2010 (Table
3.2).
Dossett et al. (2008) noted that progeny of a wild seedling from the Piedmont
of North Carolina near the southern edge of the range had significantly less winter
injury to floricanes than did progeny of cultivars originating from New York. This
49
was surprising, and it was suggested that this symptom may be primarily due to
Verticillium wilt rather than cold or other causes. In this study, ratings of symptoms
of Verticillium wilt injury in the fall were strongly correlated (r = 0.48, p<0.001)
with the degree of winter cane injury in the spring. When only the data from 2009,
after plants had been established in the ground, is considered this correlation is even
stronger (r = 0.61, p<0.001), suggesting that the bulk of winter cane injury observed
may have been due to Verticillium infection and that evaluating cane death in the
spring may be a useful way of scoring susceptibility to Verticillium wilt quickly on a
site where the pathogen is present. Ratings of Verticillium wilt symptoms were also
strongly negatively correlated with plant vigor (r = 0.54, p<0.001), indicating that
Verticillium wilt was a strong limiting factor on the performance of many of these
populations.
Fruit weight in the wild populations examined ranged from 0.5 g to 1.4 g in
2009 and 0.5 to 1.1 g in 2010, in the 2007 field (Table 3.2). ‘Munger’, ‘Jewel’, and
‘Mac Black’ were all considerably larger than most of the wild black raspberries
studied within the same year (Tables 3.2 and 3.3). Fruit weight of cultivars was
similar across fields, but was slightly larger in the younger planting in 2010 (Tables
3.2 and 3.3). Despite the fact that the fruit of wild black raspberry seedlings were
almost all smaller than the cultivars, a wide range of sizes exists, indicating
variability from which to select for improvement. Many black raspberry cultivars
and selections originated as wild growing plants that were selected for having
exceptionally large fruit. It is now thought that many of these could have been feral
50
cultivated plants or their seedlings, leading to little variation between these cultivars
despite apparently diverse origins (Dossett et al., 2010; Weber 2003). With this in
mind, particularly large-fruited populations (e.g. ORUS 3780, ORUS 3816, ORUS
3819, ORUS 3824, and ORUS 4130) may be the result of introgression from
cultivars or may be offspring of cultivars. However, it is important to remember that
none of the cultivars are more than a few generations removed from unimproved
wild germplasm, much like that being studied. Dossett et al. (2008) found low
heritability for fruit weight in a diallel consisting primarily of black raspberry
cultivars. However, fruit weight has been shown to be highly heritable in red
raspberry (Connor et al., 2005) and the same can probably be expected for black
raspberry given sufficient variation from which to select. With this in mind, the
variation present in these populations provides the opportunity to select for new
alleles for larger fruit, thereby offering the possibility to improve this trait. It is
interesting to note that fruit for nearly all of the R. leucodermis populations studied
by Finn et al. (2003) were larger than the R. occidentalis populations in this study,
despite the largest being 30% smaller than ‘Munger’, suggesting that R. leucodermis
may be valuable as a donor of alleles for large fruit weight when breeding black
raspberry.
Studying seedling population averages and comparing their performance to
clonal accessions can be valuable, particularly for gauging the potential of
populations for quantitatively inherited traits. This quantitative variation will be
particularly valuable in a breeding program focused on recurrent mass selection to
51
make genetic gain. Simply inherited traits that are fixed in these populations will also
show up strongly, however those which segregate can be masked in the statistical
analysis. Observations of the individual seedlings in this study suggest discrete
segregation for fruit gloss and sensitivity to Verticillium wilt, and these must be
weighed accordingly
While evaluating this germplasm by studying phenotypic population means
can be very valuable, it has other limitations as well. Tanksley and McCouch (1997)
outlined the need for development of QTL maps and genomic resources in the
evaluation of wild germplasm, saying, “New findings from genome research indicate
that there is tremendous genetic potential locked up in seed banks that can be
released only by shifting the paradigm from searching for phenotypes to searching
for superior genes with the aid of molecular linkage maps.” This would certainly be
beneficial in the germplasm studied here. While there was wide variation in many of
the traits examined, the path for improving some, like fruit weight, is made more
difficult when few of the accessions sampled appear to be superior to what is already
cultivated. The ability to identify novel alleles for important QTLs, or even to screen
germplasm for diversity to incorporate the most diverse material possible and
maximize the chances of recovering new QTLs for important traits, would streamline
the process of developing improved cultivars from this germplasm. Investment in
genomic resources for black raspberry would be of great value in determining the
best approach for utilizing the genetic diversity from this collection and others in the
future.
52
Despite all of these limitations, there is a broad degree of variation available
from which to select in wild black raspberry for breeding improved cultivars. More
importantly, populations and individuals with outstanding vigor and disease
resistance have been identified, opening the door to further study, as well as to the
development of improved cultivars needed by the industry.
References
Black, B., J. Frisby, K. Lewers, F. Takeda, and C.E. Finn. 2008. Heat unit model for
predicting bloom dates in Rubus. HortScience 43:2000-2004.
Card, F.W. 1898. Bush-Fruits. MacMillan, New York, NY.
Connor, A.M., M.J. Stephens, H.K. Hall, and P.A. Alspach. 2005. Variation and
heritabilities of antioxidant activity and total phenolic content estimated from a
red raspberry factorial experiment. J. Amer. Soc. Hort. Sci. 130:403–411.
Daubeny, H.A. 1996. Brambles. p. 109-190. In: Janick, J. and J.N. Moore (eds.),
Fruit Breeding: Vol. II, Vine and small fruit crops. Wiley, NY.
Dossett, M. 2007. Variation and heritability of vegetative, reproductive and fruit
chemistry traits in black raspberry (Rubus occidentalis L.). MS thesis, Oregon
State Univ., Corvallis.
Dossett, M., and C.E. Finn. 2010. Identification of resistance to the large raspberry
aphid in black raspberry. J. Amer. Soc. Hort. Sci. 135:438-444.
Dossett, M., and C.E. Finn. 2011. Primocane-fruiting in black raspberry (Rubus
occidentalis L.). J. Amer. Pom. Soc. 65:48-53.
Dossett, M., J. Lee, and C.E. Finn. 2008. Inheritance of phenological, vegetative, and
fruit chemistry traits in black raspberry. J. Amer. Soc. Hort. Sci. 133:408-417.
Dossett, M., N.V. Bassil, and C.E. Finn. 2010. Transferability of Rubus
microsatellite markers to black raspberry. Acta Hort. 859:103-106.
Drain, B.D. 1956. Inheritance in black raspberry species. Proc. Amer. Soc. Hort. Sci.
68:169-170.
Finn, C., K. Wennstrom, J. Link, and J. Ridout. 2003. Evaluation of Rubus
leucodermis populations from the Pacific Northwest. HortScience 38:11691172.
Fiola, J.A. and H.J. Swartz. 1994. Inheritance of tolerance to Verticillium albo-atrum
in raspberry. HortScience 29:1071-1073
Hall, H., K.E. Hummer, A. Jamieson, S. Jennings, and C. Weber. 2009. Raspberry
breeding and genetics. Plant Breeding Rev. 32:39-382.
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Hedrick, U.P. 1925. The small fruits of New York. N.Y. State Agric. Expt. Station.
J.B. Lyon Co. NY.
Hummer, K., M. Dossett, and C. Finn. 2008a. Plant collecting expedition for berry
crop species through Southeastern and Midwestern United States, June and
July 2007 Part I. USDA ARS NCGR Station Pub. Corvallis. 38 pp.
Hummer, K., M. Dossett, and C. Finn. 2008b. Plant collecting expedition for berry
crop species through Southeastern and Midwestern United States, June and
July 2007 Part II. USDA ARS NCGR Station Pub. Corvallis. 127 pp.
Jennings, D.L. 1966. The manifold effects of genes affecting fruit size and vegetative
growth in the raspberry. I. Gene L1. New Phytol. 65:176-187.
Jennings, D.L. 1988. Raspberries and blackberries: Their breeding, diseases and
growth. Academic Press, San Diego, CA.
Jennings, D.L., R.J. McNicol, and E, Brydon. 1986. In “Fourth Annual Report of the
Scottish Crop Research Institute for 1985”. P.84.
Lewis, D. 1939. Genetical studies in cultivated raspberries. I. Inheritance and linkage.
J. Genet. 38:367-379.
Nybom, H. and B.A. Schaal. 1990. DNA “fingerprints” reveal genotypic
distributions in natural populations of blackberries and raspberries (Rubus,
Rosaceae). Amer. J. Bot. 77:883-888.
Ourecky, D.K. 1975. Brambles, p. 98–129. In: Janick, J. and J.N. Moore (eds.).
Advances in fruit breeding. Purdue University Press, West Lafayette, IN.
Ourecky, D.K. and Slate, G.L. 1966. Hybrid vigor in Rubus occidentalis x Rubus
leucodermis seedlings. In “Proceedings of the 17th International Horticultural
Congress”, Abstract 277, Vol. 1.
Slate, G.L. and L.G. Klein. 1952. Black raspberry breeding. Proc. Amer. Soc. Hort.
Sci. 59:266-268.
Tanksley, S.D. and S.R. McCouch. 1997. Seed banks and molecular maps: unlocking
genetic potential from the wild. Science. 277:1063-1066.
USDA, NRCS. 2011. The PLANTS Database. National Plant Data Center, Baton
Rouge, LA USA. Available: http://plants.usda.gov/java/profile?symbol=RUOC
<Accessed 14 April 2011>
Weber, C.A. 2003. Genetic diversity in black raspberry detected by RAPD markers.
HortScience 38:269-272.
Wilhelm, S., and H.E. Thomas. 1950. Verticillium wilt of bramble fruits with special
reference to Rubus ursinus derivatives. Phytopathology 40:1103-1110.
Wilhelm, S., R.S. Bringhurst, and V. Voth. 1965. Origin of Rubus cultivars resistant
to Verticillium wilt. Phytopathology 55:731-733.
Williams, C.F. 1950. Influence of parentage in species hybridization of raspberries.
Proc. Amer. Soc. Hort. Sci. 56:149-156.
54
Table 3.1. USDA-ARS plant introduction (PI) number, breeding program cross
identification (ORUS) number, general provenance, and year planted for 109 wild black
raspberry (Rubus occidentalis L.) populations evaluated in replicated field plots in
Corvallis, OR.
ORUS
PI no.(s)
no.(s)
Provenance
Year
653296
4123
Mentone, AL
2008
653327
3779
Litchfield, CT
2007
652975
4117
Clayton, GA
2008
652976
4119
Clayton, GA
2008
653294
4120
Clayton, GA
2008
653298
4122
Dahlonega, GA
2008
652977
4121
Union County, GA
2008
653328
3780
Story County, IA
2007
653329
3781
Iroquois County, IL
2007
653331
3796
Greene County, IN
2007
653335
3800
Greene County, IN
2007
653332
3797
Hendricks County, IN
2007
653330
3794
Putnam County, IN
2007
653333
3798
Sullivan County, IN
2007
653334
3799
Vigo County, IN
2007
653336
3801
southern IN
2007
653299
4124
Bonner Springs, KS
2008
651846
4130
Minneapolis, KS
2008
651848
3802
Fayette County, KY
2007
653337
3803
Berkshire County, MA
2007
653338
3804
Berkshire County, MA
2007
653343
3811
Allegany County, MD
2007
653344
3812
Anne Arundel County, MD
2007
653341
3808
Harford County, MD
2007
653342
3810
Howard County, MD
2007
z
NA
3806
Howard County, MD
2007
653339
3805
Washington County, MD
2007
653340
3807
Washington County, MD
2007
653350
3821
Camden, ME
2007
653349
3820
East Vassalboro, ME
2007
653347
3817
Gardiner, ME
2007
653348
3819
Hallowell, ME
2007
651849
3815
Monmouth, ME
2007
55
Table 3.1. (continued)
ORUS
no.(s)
PI no.(s)
653345
3814
653346
3816
NA
4109
NA
4110
553765
3948
NA
4111
553764
3947
NA
4112
653323
4149
653321
4148
651847
4147
653351
3823
651851
3827
653354
3828
651850
3824
653353
3826
651852
3830
653356
3832
653357
3835
653358
3837
653359
3838
653310
4138
653308
4136
653305
4133
653309
4137
653307
4135
653363
3843
653362
3842
653360
3839
653361
3840
NA
3841
y
653364 – 653367 3844 – 3848x
653368
3849
NA
4107
NA
4108
653372
3854 – 3855
653373
3856
Provenance
Orono, ME
West Kennebunk, ME
Bath, MI
Benton Harbor, MI
Fred Russ State Forest, MI
Grand Ledge, MI
Oak Grove, MI
Okemos, MI
Belgrade, MN
Big Stone Lake National Wildlife Refuge, MN
Big Stone Lake State Park, MN
Cass County, MN
Dakota County, MN
Dakota County, MN
Ramsey County, MN
Ramsey County, MN
Fordland, MO
Fordland, MO
Madison County, NC
Rutherford County, NC
Rutherford County, NC
Chadron State Park, NE
Halsey, NE
North Loup State Recreation Area, NE
Valentine, NE
Victoria Springs State Recreation Area, NE
Columbia County, NY
Dutchess County, NY
Ontario County, NY
Ontario County, NY
Ontario County, NY
Yates County, NY
Clermont County, OH
Hilliard , OH
Newton Falls, OH
Centre County, PA
Centre County, PA
Year
2007
2007
2008
2008
2008
2008
2008
2008
2008
2008
2008
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2008
2008
2008
2008
2008
2007
2007
2007
2007
2007
2007
2007
2008
2008
2007
2007
56
Table 3.1. (continued)
ORUS
no.(s)
PI no.(s)
653369
3851
653370
3852
653371
3853
652971
4113
652973
4114
652974, 653292
4115 – 4116
653315
4142
653317
4144
653316
4143
653314
4141
653389
3904 – 3905
653395
3914 – 3915
653374
3857 – 3858
653375
3859 – 3863
653376
3864 – 3865
653377
3866 – 3867
653378
3868 – 3870
653379
3871
653380
3872 – 3876
653381
3877 – 3882
653384
3888 – 3891
653385
3892 – 3895
653398
3919
653396
3916
653397
3918
653382
3883
653390
3906 – 3907
653392
3909 – 3910
653393
3911
653394
3912 – 3913
653383
3884 – 3887
653386
3896 – 3899
653387 – 653388 3900 – 3903
653399
3926 – 3928
653401
3930
Provenance
Chester County, PA
Greene County, PA
Somerset County, PA
Glassy Mountain, SC
Glassy Mountain, SC
Rich Mountain, SC
Clay County State Park, SD
Newton Hills State Park, SD
Union Grove State Park, SD
Yankton, SD
Cannon County, TN
Cheatham County, TN
Davidson County, TN
Davidson County, TN
Davidson County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
Grundy County, TN
Grundy County, TN
Henderson County, TN
Unicoi County, TN
Unicoi County, TN
Van Buren County, TN
Van Buren County, TN
Van Buren County, TN
Van Buren County, TN
Van Buren County, TN
Warren County, TN
Warren County, TN
Warren County, TN
Columbia County, WI
Inwood, WV
Year
2007
2007
2007
2008
2008
2008
2008
2008
2008
2008
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
2007
57
Table 3.1. (continued)
ORUS
no.(s)
PI no.(s)
Provenance
Year
653402
3931
Preston County, WV
2007
653400
3929
Shepherdstown, WV
2007
653325
3777
Mactaquac, NB, Canada
2007
653326
3778
Simcoe, ON, Canada
2007
z
- Accessions not available through the USDA, ARS, National Genetic Resources Program.
Germplasm Resources Information Network (GRIN).
y
- Multiple PI numbers indicate separate seed lots from this location in GRIN.
x
- Multiple ORUS numbers indicate seed lots from multiple maternal clones at this location
were received; field plantings are a random mix from these seed lots.
Table 3.2. Vegetative and disease tolerance traits in 78 wild black raspberry (Rubus occidentalis L.) populations and
three cultivars grown in Corvallis, OR, planted in 2007, and evaluated from 2008 to 2010. Years are presented
separately to assist in comparison with Table 3.3.
Cane
stiffnessz
3.2
4.1
3.0
3.5
3.5
3.2
4.5
4.5
3.2
3.5
4.1
3.5
3.5
3.3
4.0
4.0
2.9
2.6
5.0
4.4
3.0
5.0
2.8
2.6
3.4
Vigor
2009y
5.1
3.3
5.8
5.8
5.5
4.2
3.4
5.0
4.5
4.7
4.6
5.8
3.9
4.4
4.1
4.2
4.8
5.2
5.1
5.1
4.7
5.8
4.0
4.6
5.0
Vigor
2010y
3.7
2.8
4.1
4.1
4.1
3.2
2.0
5.0
3.1
3.6
4.2
3.4
2.7
2.6
2.5
3.2
3.9
4.5
4.5
4.9
4.1
4.3
2.2
3.3
5.0
Verticillium
2008x
0.4
0.0
0.6
0.9
0.6
0.6
0.9
1.3
0.7
0.8
1.2
0.8
1.3
1.1
0.5
1.1
2.0
0.4
0.2
1.1
1.1
0.7
1.0
0.5
0.9
Verticillium
2009x
3.2
2.6
2.8
1.9
1.6
2.4
4.7
2.1
2.3
2.2
1.6
2.4
2.9
3.3
4.7
2.2
2.7
2.3
3.4
2.3
2.3
1.7
4.7
1.9
0.8
Winter
cane injury
2009w
0.5
1.1
2.6
2.7
2.2
2.6
3.1
3.3
1.8
3.3
2.5
2.0
2.3
3.1
3.2
2.9
2.7
2.0
3.4
4.0
3.6
2.0
2.8
2.3
1.3
Winter
cane injury
2010w
2.8
1.9
2.6
2.2
2.0
3.0
4.0
2.4
2.7
2.8
2.2
3.2
2.8
3.9
4.1
2.5
2.6
2.2
2.9
2.7
2.9
2.3
4.0
2.6
1.5
58
Plot
Jewel
Mac Black
Munger
ORUS 3777
ORUS 3778
ORUS 3779
ORUS 3780
ORUS 3781
ORUS 3794
ORUS 3796
ORUS 3797
ORUS 3798
ORUS 3799
ORUS 3800
ORUS 3801
ORUS 3802
ORUS 3803
ORUS 3804
ORUS 3805
ORUS 3806
ORUS 3807
ORUS 3808
ORUS 3810
ORUS 3811
ORUS 3812
Branch
anglez
3.3
3.6
1.8
1.3
2.5
2.2
3.1
3.2
3.0
2.4
3.6
2.4
3.2
1.8
3.0
3.1
1.7
1.7
4.4
3.2
2.5
3.4
1.7
3.0
2.1
Table 3.2. (continued)
Cane
stiffness
3.0
3.4
3.5
1.9
3.0
3.6
2.7
3.7
3.7
2.5
2.5
3.3
4.2
3.0
3.0
3.2
3.4
3.5
3.3
2.5
2.5
4.3
3.0
2.7
3.0
3.8
Vigor
2009
5.4
4.3
5.1
2.7
6.2
5.6
3.6
4.9
3.6
5.3
4.0
4.7
4.2
5.6
4.7
4.6
4.4
5.6
5.5
4.6
4.1
5.1
5.1
3.9
4.6
5.1
Vigor
2010
4.2
4.1
2.9
1.4
3.6
4.0
3.3
2.9
2.9
3.3
3.6
3.6
2.9
3.7
4.9
4.0
4.0
3.8
4.6
4.7
3.9
5.7
4.3
3.6
4.2
5.2
Verticillium
2008
0.8
1.2
1.3
2.5
1.0
0.8
1.0
1.1
1.8
1.1
2.4
1.1
0.9
1.8
0.9
1.4
1.4
0.8
1.1
1.7
1.2
1.4
1.2
1.0
1.0
0.7
Verticillium
2009
2.9
2.1
4.4
4.7
2.3
2.0
1.7
4.6
4.4
4.5
3.7
1.6
4.7
2.9
1.3
1.8
3.0
2.5
1.3
2.1
0.9
1.3
2.4
4.3
4.2
1.6
Winter
cane injury
2009
2.8
3.2
2.8
4.1
1.9
1.8
3.7
3.3
4.2
2.4
3.2
2.7
2.7
2.3
2.2
1.7
2.9
2.2
2.2
2.0
2.5
3.5
1.9
3.7
2.8
2.0
Winter
cane injury
2010
3.1
2.4
4.3
3.9
2.6
2.4
1.8
4.1
4.2
4.0
3.3
2.3
4.1
2.3
1.4
2.3
2.6
1.9
1.9
2.1
1.8
1.7
2.1
3.2
3.5
1.9
59
Plot
ORUS 3814
ORUS 3815
ORUS 3816
ORUS 3817
ORUS 3819
ORUS 3820
ORUS 3821
ORUS 3823
ORUS 3824
ORUS 3826
ORUS 3827
ORUS 3828
ORUS 3830
ORUS 3832
ORUS 3835
ORUS 3837
ORUS 3838
ORUS 3839
ORUS 3840
ORUS 3841
ORUS 3842
ORUS 3843
ORUS 3844-48
ORUS 3849
ORUS 3851
ORUS 3852
Branch
angle
2.3
2.8
2.5
1.6
2.5
2.5
2.6
3.4
3.0
1.6
1.9
2.9
3.0
3.0
2.5
2.5
3.0
2.5
3.0
3.0
3.0
3.5
3.0
2.5
3.0
3.5
Table 3.2. (continued)
Cane
stiffness
3.0
2.6
3.0
4.0
3.7
3.6
2.7
2.7
3.0
2.7
2.5
3.0
3.5
3.1
3.6
3.4
3.7
3.7
3.7
3.9
3.7
2.5
2.5
2.5
2.4
3.0
Vigor
2009
4.1
4.6
4.2
5.8
5.9
5.6
5.4
5.0
4.7
4.2
4.2
4.7
4.9
5.0
5.7
6.7
6.5
5.3
4.2
5.2
5.0
5.1
5.1
5.6
4.5
5.5
Vigor
2010
3.7
2.5
2.5
4.9
4.6
5.0
4.2
3.6
4.1
4.1
3.6
2.6
4.3
4.2
6.0
5.3
5.3
4.3
4.1
5.0
5.0
3.7
4.0
4.0
2.8
4.0
Verticillium
2008
0.8
1.0
1.0
0.1
0.2
0.6
0.3
0.7
0.4
1.0
0.3
1.0
0.5
0.6
0.6
0.4
1.3
0.9
1.0
1.6
0.5
0.7
0.8
0.2
1.1
0.7
Verticillium
2009
2.9
4.8
4.0
0.6
2.0
3.0
1.6
3.3
3.1
3.2
1.7
3.5
0.7
1.8
0.6
0.7
1.8
2.5
1.9
0.8
1.0
3.7
4.0
2.2
2.4
1.4
Winter
cane injury
2009
3.8
2.9
3.4
1.9
1.4
2.0
1.6
2.7
3.4
3.3
1.9
1.8
2.0
2.0
2.3
1.7
1.3
2.1
2.3
2.6
3.1
2.3
1.9
1.3
2.0
1.6
Winter
cane injury
2010
2.6
4.0
3.8
0.6
1.9
2.8
1.6
3.4
3.2
2.9
2.2
3.1
1.3
2.1
1.5
1.5
2.0
2.9
2.4
1.4
1.3
3.7
3.5
2.2
3.0
1.7
60
Plot
ORUS 3853
ORUS 3854-55
ORUS 3856
ORUS 3857-58
ORUS 3859-63
ORUS 3864-65
ORUS 3866-67
ORUS 3868-70
ORUS 3871
ORUS 3872-76
ORUS 3877-82
ORUS 3883
ORUS 3884-87
ORUS 3888-91
ORUS 3892-95
ORUS 3896-99
ORUS 3900-03
ORUS 3904-05
ORUS 3906-07
ORUS 3909-10
ORUS 3911
ORUS 3912-13
ORUS 3914-15
ORUS 3916
ORUS 3918
ORUS 3919
Branch
angle
3.0
3.0
2.8
3.5
3.0
3.5
2.4
2.8
2.6
2.5
2.0
3.5
3.4
2.8
3.7
3.4
3.0
3.0
3.5
3.0
3.7
2.5
2.5
2.6
3.0
3.0
Table 3.2. (continued)
Plot
ORUS 3926-28
ORUS 3929
ORUS 3930
ORUS 3931
Branch
angle
2.0
2.0
2.0
1.5
Cane
stiffness
4.0
4.0
3.0
2.6
Vigor
2009
6.0
6.0
4.8
6.0
Vigor
2010
3.3
4.5
3.1
4.6
Verticillium
2008
1.1
0.2
0.7
0.5
Verticillium
2009
2.3
1.5
3.4
1.0
Winter
cane injury
2009
2.3
2.2
2.0
1.6
Winter
cane injury
2010
2.5
2.4
3.2
1.5
Average
LSD
2.7
0.25
3.3
0.29
4.9
1.43
3.9
1.30
0.9
0.71
2.5
1.33
2.5
1.24
2.6
1.08
Significance (P)
Population (P) 0.0001
0.0001
0.0001
0.0001
0.0001
Replication
0.172
0.131
0.0001
0.0312
0.0247
Year (Y)
----0.0001
0.0001
0.0119
P×Y
----0.0001
0.0001
0.0001
z
– Branch angle and cane stiffness rated on a subjective scale from 1-5, where 1 = least stiff and ~75o-90o angle and 5 =
most stiff and < 15o angle.
y
– Primocane vigor scored on a subjective scale from 1-9, where 1 = least vigorous and 9 = most vigorous.
x
– Verticillium symptoms scored on a scale from 0-6, where 0 = no apparent Verticillium symptoms and 6 = all
primocanes showing discolored and stunting.
w
– Winter cane injury scored on a subjective scale from 0-5, where 0 = no winter cane injury to canes or cane tips and 5 = 80%100% of floricanes dead.
61
Table 3.3. Vegetative and disease tolerance traits in 31 wild black raspberry (Rubus occidentalis L.) populations and three
cultivars grown in Corvallis, OR, planted in the spring of 2008 and evaluated from 2008 to 2010. Years are presented
separately to assist in comparison with Table 3.2.
Branch
anglez
3.0
3.4
1.7
2.0
1.7
1.8
1.6
2.0
2.9
2.8
2.0
2.3
2.6
4.5
3.9
3.0
3.5
3.0
3.2
3.5
3.0
3.0
1.7
1.4
1.0
Cane
stiffnessz
3.0
4.3
3.0
2.4
1.7
2.0
2.2
3.0
2.6
2.3
2.0
3.0
3.8
4.8
4.0
3.6
4.1
3.6
4.3
2.8
3.0
3.0
1.5
2.0
2.0
Vigor
2009y
5.5
4.7
5.8
4.5
3.4
4.3
4.2
4.2
4.1
4.4
4.6
5.2
6.1
6.5
5.4
5.7
5.6
6.3
5.5
5.2
3.8
2.9
2.6
2.3
3.2
Vigor
2010y
4.2
3.8
4.3
3.8
3.1
3.5
3.7
3.4
3.7
4.4
3.8
4.3
3.9
5.8
4.2
4.3
4.2
5.0
5.5
4.3
2.9
2.9
2.3
2.2
3.3
Verticillium
2008x
0.0
0.8
0.5
1.7
0.6
1.0
0.9
0.4
NA
0.4
0.3
0.6
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.2
NAv
NA
NA
NA
NA
Verticillium
2009x
3.2
3.1
3.2
3.6
5.0
3.5
3.4
3.5
2.5
3.5
3.0
3.3
4.4
0.2
3.0
3.0
3.0
2.0
2.1
2.1
3.7
3.2
3.9
4.4
2.8
Winter cane
injury 2009w
2.9
3.5
3.5
3.3
4.3
3.4
3.2
3.1
2.8
3.4
2.7
3.0
3.5
0.6
2.5
2.6
2.3
1.9
2.1
1.9
3.2
2.6
3.6
3.9
3.1
62
Plot
Jewel
Mac Black
Munger
ORUS 3947
ORUS 3948
ORUS 4107
ORUS 4108
ORUS 4109
ORUS 4110
ORUS 4111
ORUS 4112
ORUS 4113
ORUS 4114
ORUS 4115-16
ORUS 4117
ORUS 4119
ORUS 4120
ORUS 4121
ORUS 4122
ORUS 4123
ORUS 4124
ORUS 4130
ORUS 4133
ORUS 4135
ORUS 4136
Table 3.3. (continued)
Plot
ORUS 4135
ORUS 4136
ORUS 4137
ORUS 4138
ORUS 4141
ORUS 4142
ORUS 4143
ORUS 4144
ORUS 4147
ORUS 4148
ORUS 4149
Branch
anglez
1.4
1.0
1.0
1.2
2.3
2.2
2.3
1.4
1.7
1.0
1.4
Cane
stiffnessz
2.0
2.0
1.0
1.6
1.0
1.0
1.6
1.7
1.7
1.0
1.8
Vigor
2009y
2.3
3.2
1.3
3.0
2.3
3.0
3.1
3.1
3.0
2.4
3.8
Vigor
2010y
2.2
3.3
1.8
3.3
2.4
2.4
2.9
2.8
3.1
2.8
4.2
Verticillium
2008x
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Verticillium
2009x
4.4
2.8
5.2
1.0
5.1
4.1
4.1
3.5
4.6
3.9
3.5
Winter cane
injury 2009w
3.9
3.1
3.2
1.7
4.2
3.6
3.4
3.0
3.4
3.0
3.2
Average
LSD
2.3
0.23
2.5
0.32
4.1
0.90
3.6
1.17
0.4
0.42
3.3
1.55
3.0
1.36
Significance (P)
Population (P) 0.0001
0.0001
0.0001
0.0001
0.0006
Replication
0.244
0.184
0.042
0.370
0.4433
Year (Y)
----0.0001
0.0001
--P×Y
----0.033
0.019
--z
– Branch angle and cane stiffness rated on a subjective scale from 1-5, where 1 = least stiff and ~75o-90o angle and 5 = most
stiff and < 15o angle.
y
– Primocane vigor scored on a subjective scale from 1-9, where 1 = least vigorous and 9 = most vigorous.
x
– Verticillium symptoms scored on a scale from 0-6, where 0 = no apparent Verticillium symptoms and 6 = all primocanes
showing discolored and stunting.
w
– Winter cane injury scored on a subjective scale from 0-5, where 0 = no winter cane injury to canes or cane tips and 5 = 80%-100% of
floricanes dead.
v
- Unable to assess due to small plant size in the establishment year.
63
Table 3.4. Phenology and fruiting traits in 78 wild black raspberry (Rubus occidentalis L.) populations and three cultivars
grown in Corvallis, OR planted in 2007 and evaluated in 2009 and 2010. Years are presented separately to assist in
comparison with Table 3.5.
Flowering
date 2009
24-May
4-Jun
19-May
21-May
17-May
22-May
21-May
19-May
21-May
22-May
19-May
17-May
21-May
17-May
1-Jun
20-May
18-May
19-May
22-May
21-May
22-May
20-May
18-May
22-May
17-May
19-May
Flowering
date 2010
21-May
8-Jun
14-May
17-May
15-May
19-May
24-May
14-May
17-May
18-May
17-May
13-May
17-May
14-May
6-Jun
16-May
16-May
13-May
17-May
14-May
16-May
17-May
17-May
15-May
13-May
15-May
Ripening
date 2009
28-Jun
7-Jul
22-Jun
26-Jun
23-Jun
23-Jun
28-Jun
25-Jun
26-Jun
25-Jun
24-Jun
22-Jun
24-Jun
20-Jun
8-Jul
21-Jun
21-Jun
23-Jun
25-Jun
22-Jun
24-Jun
24-Jun
21-Jun
24-Jun
21-Jun
24-Jun
Ripening
date 2010
1-Jul
14-Jul
28-Jun
28-Jun
27-Jun
30-Jun
6-Jul
26-Jun
1-Jul
28-Jun
3-Jul
24-Jun
28-Jun
26-Jun
13-Jul
30-Jun
27-Jun
23-Jun
30-Jun
29-Jun
30-Jun
3-Jul
28-Jun
28-Jun
29-Jun
29-Jun
Fruit weight
2009 (g)
2.3
1.9
1.3
0.8
0.8
0.8
1.3
0.7
0.7
0.7
0.7
0.9
0.9
0.9
0.9
0.7
0.8
0.7
0.9
0.7
0.7
0.8
0.8
1.0
0.6
0.7
Fruit weight
2010 (g)
1.5
1.3
1.0
0.7
0.7
0.7
1.1
0.6
0.6
0.6
0.8
0.8
0.7
0.7
0.5
0.6
0.6
0.6
0.6
0.7
0.7
0.7
0.7
0.7
0.6
0.6
Fruit gloss
2009z
3.0
2.7
3.1
3.3
3.0
2.6
3.5
2.5
3.6
2.7
2.4
2.4
3.8
4.8
2.7
3.1
2.7
3.0
2.2
3.8
2.7
3.1
3.5
2.2
4.4
2.4
Fruit gloss
2010z
3.4
2.5
3.3
3.4
3.3
2.8
3.7
3.5
4.2
3.9
3.2
2.7
3.6
4.6
3.0
3.2
3.3
3.5
2.8
4.2
2.6
3.0
4.0
2.8
4.0
3.3
64
Population
Jewel
Mac Black
Munger
ORUS 3777
ORUS 3778
ORUS 3779
ORUS 3780
ORUS 3781
ORUS 3794
ORUS 3796
ORUS 3797
ORUS 3798
ORUS 3799
ORUS 3800
ORUS 3801
ORUS 3802
ORUS 3803
ORUS 3804
ORUS 3805
ORUS 3806
ORUS 3807
ORUS 3808
ORUS 3810
ORUS 3811
ORUS 3812
ORUS 3814
Table 3.4. (continued)
Flowering
date 2009
21-May
20-May
21-May
19-May
20-May
21-May
19-May
20-May
16-May
17-May
16-May
20-May
18-May
19-May
24-May
22-May
19-May
18-May
18-May
19-May
15-May
20-May
21-May
22-May
21-May
24-May
22-May
Flowering
date 2010
16-May
14-May
17-May
13-May
25-May
17-May
13-May
14-May
7-May
11-May
11-May
14-May
13-May
17-May
20-May
22-May
16-May
16-May
12-May
14-May
20-May
16-May
13-May
13-May
27-May
19-May
23-May
Ripening
date 2009
24-Jun
24-Jun
25-Jun
22-Jun
24-Jun
24-Jun
23-Jun
23-Jun
19-Jun
22-Jun
20-Jun
24-Jun
23-Jun
23-Jun
28-Jun
25-Jun
25-Jun
22-Jun
22-Jun
23-Jun
24-Jun
27-Jun
22-Jun
25-Jun
26-Jun
27-Jun
28-Jun
Ripening
date 2010
2-Jul
29-Jun
29-Jun
26-Jun
5-Jul
29-Jun
25-Jun
26-Jun
24-Jun
26-Jun
27-Jun
30-Jun
27-Jun
28-Jun
7-Jul
5-Jul
30-Jun
27-Jun
27-Jun
27-Jun
1-Jul
29-Jun
26-Jun
25-Jun
7-Jul
29-Jun
1-Jul
Fruit weight
2009 (g)
0.6
1.0
0.6
1.1
0.9
0.6
0.9
1.4
0.6
0.5
0.6
0.8
0.8
1.0
0.7
0.6
0.9
0.8
0.7
0.6
0.8
1.0
0.9
0.7
0.9
0.8
0.7
Fruit weight
2010 (g)
0.6
0.8
0.6
0.9
0.7
0.6
0.9
1.0
0.6
0.6
0.6
0.7
0.6
0.9
0.6
0.6
0.8
0.7
0.6
0.7
0.6
0.7
0.7
0.7
0.8
0.6
0.8
Fruit gloss
2009z
3.3
2.7
2.5
4.1
3.0
2.7
2.6
3.0
3.9
3.1
2.8
3.0
3.4
2.7
2.9
4.8
3.2
2.3
3.7
4.5
3.1
2.8
3.6
1.7
4.1
3.3
2.2
Fruit gloss
2010z
4.1
3.3
3.5
4.0
3.6
3.2
2.8
2.7
3.5
3.5
3.3
3.4
3.4
3.7
3.2
4.6
3.5
3.0
4.1
4.2
3.5
3.4
4.3
2.7
3.5
4.3
3.1
65
Population
ORUS 3815
ORUS 3816
ORUS 3817
ORUS 3819
ORUS 3820
ORUS 3821
ORUS 3823
ORUS 3824
ORUS 3826
ORUS 3827
ORUS 3828
ORUS 3830
ORUS 3832
ORUS 3835
ORUS 3837
ORUS 3838
ORUS 3839
ORUS 3840
ORUS 3841
ORUS 3842
ORUS 3843
ORUS 3844-48
ORUS 3849
ORUS 3851
ORUS 3852
ORUS 3853
ORUS 3854-55
Table 3.4. (continued)
Flowering
date 2009
21-May
20-May
19-May
20-May
22-May
23-May
24-May
24-May
22-May
22-May
23-May
20-May
21-May
19-May
22-May
18-May
19-May
21-May
23-May
18-May
20-May
22-May
19-May
20-May
18-May
17-May
20-May
21-May
Flowering
date 2010
13-May
11-May
12-May
14-May
13-May
19-May
20-May
20-May
24-May
17-May
14-May
13-May
14-May
14-May
15-May
12-May
13-May
16-May
18-May
14-May
16-May
16-May
12-May
22-May
15-May
12-May
17-May
20-May
Ripening
date 2009
24-Jun
24-Jun
23-Jun
24-Jun
26-Jun
26-Jun
26-Jun
24-Jun
24-Jun
25-Jun
26-Jun
23-Jun
22-Jun
20-Jun
23-Jun
22-Jun
21-Jun
26-Jun
26-Jun
22-Jun
25-Jun
23-Jun
21-Jun
22-Jun
25-Jun
22-Jun
23-Jun
26-Jun
Ripening
date 2010
29-Jun
26-Jun
26-Jun
1-Jul
27-Jun
2-Jul
2-Jul
30-Jun
1-Jul
2-Jul
29-Jun
28-Jun
22-Jun
27-Jun
29-Jun
24-Jun
27-Jun
28-Jun
29-Jun
28-Jun
28-Jun
28-Jun
28-Jun
1-Jul
29-Jun
18-Jun
30-Jun
2-Jul
Fruit weight
2009 (g)
0.7
0.9
0.8
0.7
0.8
0.7
0.8
0.7
0.8
1.0
0.6
0.8
0.8
1.0
0.9
0.6
0.7
0.6
0.6
0.9
0.7
0.9
1.0
0.8
0.8
0.7
0.9
0.8
Fruit weight
2010 (g)
0.6
0.8
0.7
0.7
0.7
0.6
0.7
0.6
0.8
0.7
0.6
0.6
0.8
0.7
0.8
0.7
0.6
0.7
0.6
0.7
0.7
0.8
0.8
0.7
0.7
0.6
0.9
0.7
Fruit gloss
2009z
3.4
4.1
3.7
3.6
4.0
4.3
4.2
2.1
2.6
2.3
3.3
3.4
4.0
3.6
3.9
3.0
3.8
3.3
2.3
2.9
3.5
3.5
3.5
3.8
3.1
3.5
2.3
2.9
Fruit gloss
2010z
3.7
4.0
4.2
3.7
4.7
4.4
3.8
2.4
3.6
2.9
3.6
3.9
4.4
3.2
4.3
4.0
4.3
3.7
2.6
3.8
3.4
4.0
4.2
4.2
3.7
3.5
2.6
3.2
66
Population
ORUS 3856
ORUS 3857-58
ORUS 3859-63
ORUS 3864-65
ORUS 3866-67
ORUS 3868-70
ORUS 3871
ORUS 3872-76
ORUS 3877-82
ORUS 3883
ORUS 3884-87
ORUS 3888-91
ORUS 3892-95
ORUS 3896-99
ORUS 3900-03
ORUS 3904-05
ORUS 3906-07
ORUS 3909-10
ORUS 3911
ORUS 3912-13
ORUS 3914-15
ORUS 3916
ORUS 3918
ORUS 3919
ORUS 3926-28
ORUS 3929
ORUS 3930
ORUS 3931
Table 3.4. (continued)
Population
Average
LSD
Flowering
date 2009
20-May
4.15 (days)
Flowering Ripening
date 2010 date 2009
16-May
24-Jun
2.66 (days) 3.87 (days)
Ripening
date 2010
29-Jun
2.67 (days)
Fruit weight
2009 (g)
0.8
0.22
Fruit weight
2010 (g)
0.7
0.15
Significance (P)
Population (P)
0.0001
0.0001
0.0001
Replication
0.1118
0.0001
0.207
Year (Y)
0.0001
0.0001
0.0001
P×Y
0.0821
0.1374
0.051
z
- Fruit gloss scored on a subjective scale, where 1 = most pubescent and 5 = most glossy.
Fruit gloss
2009z
3.2
0.92
Fruit gloss
2010z
3.5
0.64
0.0001
0.159
0.0001
0.0643
67
68
Table 3.5. Phenology and fruiting traits in 31 wild black raspberry (Rubus
occidentalis L.) populations and three cultivars grown in Corvallis, OR, planted in
2008 and evaluated in 2010.
Pop
Jewel
Mac Black
Munger
ORUS 3947
ORUS 3948
ORUS 4107
ORUS 4108
ORUS 4109
ORUS 4110
ORUS 4111
ORUS 4112
ORUS 4113
ORUS 4114
ORUS 4115-16
ORUS 4117
ORUS 4119
ORUS 4120
ORUS 4121
ORUS 4122
ORUS 4123
ORUS 4124
ORUS 4130
ORUS 4133
ORUS 4135
ORUS 4136
ORUS 4137
ORUS 4138
ORUS 4141
ORUS 4142
ORUS 4143
ORUS 4144
ORUS 4147
ORUS 4148
ORUS 4149
Flowering
date 2010
24-May
8-Jun
18-May
21-May
15-May
24-May
23-May
24-May
23-May
23-May
24-May
23-May
21-May
25-May
24-May
17-May
26-May
23-May
23-May
25-May
25-May
27-May
24-May
21-May
21-May
24-May
18-May
23-May
22-May
22-May
20-May
20-May
23-May
14-May
Ripening date
2010
1-Jul
15-Jul
27-Jun
25-Jun
25-Jun
1-Jul
3-Jul
2-Jul
30-Jun
29-Jun
25-Jun
27-Jun
29-Jun
5-Jul
29-Jun
25-Jun
27-Jun
30-Jun
3-Jul
30-Jun
8-Jul
5-Jul
28-Jun
27-Jun
25-Jun
28-Jun
25-Jun
27-Jun
27-Jun
24-Jun
27-Jun
26-Jun
28-Jun
23-Jun
Fruit weight
2010(g)
1.7
1.6
1.2
0.7
0.3
0.8
0.8
0.9
1.0
0.7
0.7
0.8
0.6
0.9
0.6
0.6
0.5
0.7
0.8
0.7
1.0
1.2
0.6
0.7
0.6
0.6
0.6
0.5
0.8
0.6
0.6
0.7
0.6
0.6
Fruit gloss
2010z
3.8
2.7
3.7
3.2
1.9
3.8
3.6
3.7
3.7
4.3
3.9
4.5
3.4
2.9
2.7
1.9
3.1
3.0
3.3
3.6
3.0
4.1
4.3
2.5
3.6
3.8
3.7
2.6
3.1
3.4
1.9
2.9
2.5
2.8
Average
LSD
23-May
3.3 (days)
29-Jun
1.9 (days)
0.8
0.22
3.2
0.67
Significance (P)
Population
0.0001
0.0001
0.0001
0.0001
Replication
0.910
0.96
0.019
0.236
z
- Fruit gloss scored on a subjective scale, where 1 = most pubescent and 5 = most glossy.
69
Table 3.6. Breeding program population identification (ORUS) number, state of origin, and
primocane-fruiting status of primocane-fruiting seedlings in wild black raspberry (Rubus
occidentalis L.) germplasm collected in 2006 and 2007 and evaluated in the field in
Corvallis, Oregon from 2008 to 2010.
ORUS number
ORUS 3779
ORUS 3797
ORUS 3798
ORUS 3801
ORUS 3803
ORUS 3811
ORUS 3812
ORUS 3814
ORUS 3816
ORUS 3817
ORUS 3821
ORUS 3823
ORUS 3824
ORUS 3826
ORUS 3839
ORUS 3840
ORUS 3843
ORUS 3852
ORUS 3859-63
ORUS 3864-65
ORUS 3866-67
ORUS 3910
ORUS 3912
ORUS 3926-28
ORUS 3931
ORUS 4110
ORUS 4112
ORUS 4113
ORUS 4114
ORUS 4115-16
ORUS 4117
ORUS 4119
ORUS 4130
ORUS 4135
State of origin
CT
IN
IN
IN
MA
MD
MD
ME
ME
ME
ME
MN
MN
MN
NY
NY
NY
PA
TN
TN
TN
TN
TN
WI
WV
MI
MI
SC
SC
SC
GA
GA
KS
NE
Year primocane-fruiting was observed
2008
2009
2010
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
70
Fig 3.1. Variation in fruit size, color, and gloss, in seedlings of wild Rubus occidentalis L. populations grown in Corvallis,
OR. From left to right, top row: ORUS 3778, Simcoe, Ontario; ORUS 3778, Simcoe, Ontario; ORUS 3857, Davidson County,
Tennessee. Bottom row: ORUS 4115, Rich Mountain, South Carolina; ORUS 4149, Cincinnati, Ohio; ‘Munger’.
71
Fig. 3.2. Leaves from phenotypically normal Rubus occidentalis L. leaves, with sessile leaflets (left), and leaves from a
seedling of ORUS 3841, showing leaflets attached to the main rachis by petiolules.
A
B
C
D
Fig 3.3. Flowering abnormalities segregating in ORUS 3801. A, showing elongated leafy sepals on closed flower; B, showing
one of five elongated and reflexed stamens in place of petals; C, showing large leafy sepals and elongated stamen; D, normal
R. occidentalis L. flower.
72
73
Chapter 4:
Characterization of a novel anthocyanin profile in wild black
raspberry mutants: an opportunity for studying the genetic control
of pigment and color
Michael Dossett, Jungmin Lee, and Chad E. Finn
Journal of Functional Foods
P.O. Box 29095,
12 Gleneyre St.
St. John’s, NL A1A 5B5
Canada
Accepted (In Press)
74
Abstract
The type and amount of anthocyanins in raspberries, and other small fruits,
has recently received increased attention. Black raspberry (Rubus occidentalis L.), in
particular, has long been recognized as a rich source of anthocyanins and has been
the focus of many recent studies examining their potential health benefits. In this
study, we characterized a novel anthocyanin profile found in seedlings of two wild
black raspberry populations collected from South Dakota, USA. Seedlings from
these populations lack pigments glycosylated with rutinoside in their fruit, have
elevated levels of cyanidin-3-sambubioside, and contain a small but significant
amount of pelargonidin-3-glucoside, a pigment reported only once previously in
black raspberry. Affected fruit also have lower than typical total anthocyanins (77.5
to 134.4 mg·100 mL-1). Based on the available evidence, we believe the plants have
a mutation in the gene encoding anthocyanidin-3-glycoside rhamnosyltransferase
(3RT), providing a unique opportunity to identify and study one of the major genes
in the anthocyanin pathway and its effect on fruit anthocyanins and color.
Introduction
In addition to traditional markets for consumption of fresh and processed
black raspberry fruit, there is a long history of its use as a natural colorant and dye
because of its high anthocyanin levels (Hong and Wrolstad, 1990a; Lee and Slate,
1954). Studies characterizing the types of anthocyanins present in black raspberry
75
fruit date back to at least the 1960s (Nybom, 1968). A number of recent studies have
examined the anthocyanin composition of black raspberry fruit using more
sophisticated tools than those available 50 years ago and have consistently detected
cyanidin-3-glucoside, cyanidin-3-sambubioside, cyanidin-3-rutinoside, cyanidin-3xylosylrutinoside, pelargonidin-3-rutinoside, and peonidin-3-rutinoside (Dossett et
al., 2008; Tian et al., 2006a, 2006b; Tulio, et al., 2008; Wyzgoski et al., 2010). Wu et
al. (2006) also found trace levels of pelargonidin-3-glucoside in black raspberry fruit.
The anthocyanins in black raspberry fruit are comprised of three anthocyanin
aglycones: cyanidin, pelargonidin, and peonidin, glycosylated with various
combinations of three different sugars: glucose, rhamnose, and xylose. Anthocyanin
biosynthesis has been well studied in a variety of plants and the biosynthetic pathway
appears to be heavily conserved (Holton and Cornish, 1995). The six major
anthocyanin aglycones are pelargonidin, cyanidin, peonidin, delphinidin, petunidin,
and malvidin. Cyanidin-, pelargonidin-, and delphinidin-glucosides are the base
anthocyanins, which then may undergo further glycosylation and/or B-ring
modification (to produce petunidin, peonidin, and malvidin) in a series of stepwise
modifications (Tanaka et al., 2008).
The anthocyanins of black raspberries, and other highly pigmented small
fruits, have received increased attention in recent years because of interest in their
potential health benefits. Black raspberries and other sources of dietary anthocyanins
have been linked to many possible health benefits such as reducing eyestrain,
improving night vision, helping to prevent macular degeneration, anti-inflammatory
76
effects, protecting against DNA damage, and exhibiting anti-cancer activities (Afaq
et al., 2005; Kresty et al., 2001, 2006; Lazze et al., 2003; Wang et al., 1999), and
have been well reviewed (de Pascual-Teresa and Sanchez-Ballesta, 2008; Espin et al.,
2007; Rao and Snyder, 2010). Studies linking the high levels of anthocyanins in
black raspberry with potential health benefits have led to increasing interest in black
raspberry from consumers and from various functional food and nutraceutical
markets (Espin et al., 2007).
Black raspberry fruit is dominated by cyanidin-3-rutinoside and cyanidin-3xylosylrutinoside, which account for 80% or more of the total anthocyanins (Dossett
et al., 2010; ; Hong and Wrolstad, 1990a; Ozgen et al., 2008; Tian et al., 2006a,
2006b; Tulio, et al., 2008; Wyzgoski et al., 2010). These two main anthocyanins are
also more potent phenolic antioxidants (cyanidin-3-xylosylrutinoside > cyanidin-3rutinoside) compared to the other anthocyanins present in black raspberry fruit (Tulio
et al., 2008), though limited information is available about the potential bioactivity of
individual cyanidin-based anthocyanins with different sugar moieties (Stintzing et al.,
2002; Stoner et al., 2005; Tian et al., 2006b; Tulio et al., 2008) or their relative
desirability for product development, food processing, natural colorant usage, and
storability (Hager et al., 2008; Hong and Wrolstad, 1990a; Stintzing et al., 2002).
Aside from interest in their potential health benefits, anthocyanins in red and
black raspberry play a more basic role as an indicator of fruit quality, suitability for
different markets, and ultimately consumer acceptance. Fruit color is a function of
not just the total amount but also the type of anthocyanins present, with different
77
aglycones, glycosylation, and acylation each contributing to the overall color of the
fruit in addition to pH and interactions with other fruit components (CastañedaOvando et al., 2009; ; Giusti et al., 1999; Stintzing et al., 2002; Tanaka et al., 2008).
In black and red raspberry, color is a critical indicator of quality and suitability for
processing with darker fruit generally being preferred over lighter colored fruit (Hall
et al., 2009). Studies have shown consumer perceptions of fruit freshness, ripeness,
and flavor to be heavily influenced by color (Garber et al., 2000; Hall et al., 2009;
Zampini et al., 2007; Zellner and Durlach, 2003), underscoring the importance of
color and color stability for fresh market fruit as well.
Despite the interest in black raspberry anthocyanins, little is known about the
genetic control and regulation of their production in the fruit. While many of the
enzymes involved in anthocyanin biosynthesis have been identified in model plant
systems (Petunia × hybrida Hort., Antirrhinum majus L., and Zea mays L.), none of
the genes involved in anthocyanin production have been mapped or identified in
black raspberry. This is made difficult by the fact that, while anthocyanin deficient
(yellow/orange-fruited) mutants have been recognized for more than a century (Card,
1898) and differences in the relative proportion of anthocyanins have been reported
(Dossett et al., 2008, 2010), little variation in the types of anthocyanins present in
black raspberry fruit has been found. While a major effort to identify and map genes
involved in anthocyanin biosynthesis and fruit color is underway in red raspberry
(Kassim et al., 2009; McCallum et al., 2010), the apparent lack of variation in
cultivated black raspberry has limited similar work in this crop. The objective of this
78
study was to identify black raspberry seedlings, collected from wild populations, that
might contain a novel anthocyanin profile, and to examine their potential for use in
further studies on the genetic control of black raspberry anthocyanin biosynthesis.
Materials and methods
Plant materials and sample preparation. Seeds of ORUS 4141 and ORUS 4143 were
collected in July 2007 from wild black raspberry plants at Lewis and Clark State
Park (Yankton, SD, USA) and Union Grove State Park (Beresford, SD, USA),
respectively. These two collection sites are about 90 km apart. The seed was
scarified, stratified and germinated the following fall in the greenhouse using
standard protocols (Finn and Hancock, 2008) and seedlings planted in the field in
Corvallis, OR (USA) in May 2008 along with seedlings from 30 other wild black
raspberry populations and the cultivars Munger, Jewel, and Mac Black in a
randomized complete block design with four replicated plots of four plants each.
Details of the field maintenance are described in Dossett et al. (2008). This project
was part of a broader research effort to widen the genetic base of cultivated black
raspberry.
Fruit were harvested from this plot in July 2010 for analysis of anthocyanins
and other maturity traits (data not shown). Protocols used for fruit harvest and
sample preparation were similar to those previously described (Dossett et al., 2008,
2010). Briefly, 25 berries from each plant (genotype) were picked, weighed, and
79
added to a bulk fruit sample of the population for each plot. Due to disease
(primarily Verticillium wilt [Verticillium dahliae Kleb. or V. albo-atrum Reinke and
Berthold]), only three of the four replicated plots of ORUS 4141 and ORUS 4143
had sufficient fruit for analysis (Table 4.1). As mentioned previously, this research
was part of a larger project to identify useful wild germplasm for widening the
genetic base of cultivated black raspberry; fruit from the 30 other wild populations
were collected and analyzed, but fall outside the scope of this study, which focuses
on ORUS 4141 and ORUS 4143. An entire replication (rep) was picked in a single
day to minimize variation from the effects of differing irrigation status and weather
at harvest. Harvested fruit was packed on ice immediately after harvest and then
frozen immediately (–23 oC) after arrival at the laboratory. Bulked samples were
thawed and extracted as described by Dossett et al. (2008). An aliquot of each
sample was centrifuged at room temperature at 2547 gn for 20 min to separate the
juice from the pulp. The supernatant was then diluted with HPLC grade water, and
filtered with a 0.45 µm syringe driven Millex-FH filter (Millipore, Bedford, MA,
USA) prior to further analysis.
Analysis of anthocyanins. Total anthocyanins were determined by HPLC by
summing the amounts of the individual anthocyanins detected. Anthocyanin profiles
were determined by HPLC/diode array detector/ion trap XCT mass spectrometer
(HPLC/DAD/ESI-MS/MS) on an Agilent 1100 series system (Agilent Technologies,
Santa Clara, CA, USA). The guard and analytical columns, mobile phase
80
composition, and the gradient program used for HPLC analysis are described by Lee
and Finn (2007). Sample injection volume was 5 µL. Anthocyanins were monitored
at 520 nm and quantified with a cyanidin-3-glucoside standard (Polyphenol As,
Sandnes, Norway). Frozen strawberries (grown in USA, distributed by Western
Family Foods Inc., Portland, OR, USA) were purchased at a local marketplace
(Parma, ID, USA), extracted, filtered, and injected onto the HPLC/DAD/ESIMS/MS to confirm the identity of pelargonidin-3-glucoside (Hong and Wrolstad,
1990b). Ultraviolet-visible (UV-Vis) absorption spectra (190 to 600 nm) were
collected for all peaks. ESI-MS/MS parameters were set as described in Lee and
Finn (2007). Individual peak assignments were made according to UV-Vis spectra,
retention times, molecular ions mass-to-charge ratio (m/z), and fragmented ions m/z.
Quantification was performed with HPLC/DAD results.
Results and discussion
Of the six samples from ORUS 4141 and ORUS 4143, only one (ORUS 4141,
rep 2) had an anthocyanin profile closely resembling previous work under identical
analytical conditions (Dossett et al., 2008, 2010). Six anthocyanins were detected in
ORUS 4141, rep 2 (in elution order): cyanidin-3-sambubioside, cyanidin-3-glucoside,
cyanidin-3-xylosylrutinoside, cyanidin-3-rutinoside, pelargonidin-3-rutinoside, and
peonidin-3-rutinoside, with cyanidin-3-glucoside and cyanidin-3-xylosylrutinoside
co-eluting (Table 4.1, Fig. 4.1). The relative proportions of these six anthocyanins
81
were also similar to values previously reported for cultivated black raspberry
(Dossett et al., 2010) with cyanidin-3-glucoside and cyanidin-3-xylosylrutinoside
combining for approximately 67% of the total anthocyanins and cyanidin-3rutinoside comprising another 24%. A second sample from this same population
(ORUS 4141, rep 4) had a similar anthocyanin profile, containing the same six
anthocyanins, as well as small amounts of pelargonidin-3-glucoside. This sample
was also noted for having an elevated proportion of cyanidin-3-sambubioside and
much lower total anthocyanins (Table 4.1). The remaining sample (rep 2) from
ORUS 4141 and all three samples (reps 1, 2, and 3) from ORUS 4143 had
significantly altered anthocyanin profiles (Fig. 4.1) with only three anthocyanins
detected: cyanidin-3-sambubioside, cyanidin-3-glucoside, and pelargonidin-3glucoside. In addition to their notable lack of rhamnose containing anthocyanins,
these four samples were also characterized by elevated proportions of cyanidin-3sambubioside and relatively low total anthocyanins (Table 4.1).
While this is the first report of black raspberry fruit lacking anthocyanidin-3rutinosides, variation for presence/absence of rutinoside pigments has been noted in
red raspberry. Francis (1972), Misic (1973), and Barritt and Torre (1975a) noted
variation in the presence of cyanidin-3-rutinoside and pelargonidin-3-rutinoside in
several red raspberry varieties. Barritt and Torre (1975b) studied the inheritance of
rhamnose-containing pigments in red raspberry and concluded that it was controlled
by a dominant gene, which they designated R. They also found segregation for
xylose-containing pigments in red raspberry, which was further supported by
82
Jennings and Carmichael (1980) who designated the dominant gene controlling this
trait as Xy. While gene R occurs in a range of red raspberry germplasm, gene Xy is
more unusual in red raspberry and probably was introduced to red raspberry through
inter-specific hybridization with black raspberry for other traits (Jennings and
Carmichael, 1980).
The complete absence of anthocyanidin-3-rutinosides, but not their
precursors, in fruit of some black raspberry genotypes is strong evidence for
homozygous mutant or null alleles at the locus corresponding to gene R described in
red raspberry (Barritt and Torre, 1975b). Kamsteeg et al. (1979) identified UDPrhamnose anthocyanidin-3-glucoside rhamnosyltransferase (3RT) as the enzyme
responsible for catalyzing the addition of rhamnose to the 6- position of the glucose
bound at the 3- position of the anthocyanidin skeleton, thereby forming
anthocyanidin-3-rutinosides from anthocyanidin-3-glucosides in Silene L. Brugliera
et al. (1994) and Kroon et al. (1994) sequenced and cloned the gene for 3RT in P. ×
hybrida, and confirmed that it was responsible for catalyzing the formation of
anthocyanidin-3-rutinosides. The absence of fruit with normal pigment composition
in all samples from Union Grove State Park (ORUS 4143) suggests that a nonfunctional 3RT allele (r) is fixed in this population. The data also indicate that this
allele segregated in the population from Lewis and Clark State Park (ORUS 4141).
One sample from Lewis and Clark State Park closely resembled the three from
Union Grove State Park, while another contained the normal pigment profile
described in black raspberry. The composition of the third sample from Lewis and
83
Clark State Park (ORUS 4141, rep 4) with pelargonidin-3-glucoside in addition to
the expected anthocyanins, along with its lower total anthocyanin concentration and
elevated proportion of cyanidin-3-sambubioside indicates that this is likely a mixed
sample, representing bulked fruit from normal (RR or Rr) and homozygous recessive
(rr) plants.
Schram et al. (1984) and Tornielli et al. (2009) noted that homozygous
mutations in 3RT in P. × hybrida resulted in the accumulation of anthocyanin-3glucosides. It is difficult to confirm this result in the present black raspberry study,
because cyanidin-3-glucoside could not be quantified in fruit from normally
pigmented plants as a result of coelution with cyanidin-3-xylosylrutinoside. However,
based on an extracted ion chromatogram of 449, only minor amounts of cyanidin-3glucoside were detected in ORUS 4141, rep 3. It also appears that anthocyanidin-3glucosides did not accumulate to a large degree in the fruit from rr plants, and
certainly not by the amount expected to account for the missing proportions of
cyanidin-3-rutinoside and its derivatives. The single normal sample in this study
(ORUS 4141, rep 3) contained 103.5 mg·100 mL-1 of cyanidin-3-rutinoside. This is
only slightly less than the range of 113.6 to 257.3 mg·100 mL-1 found by Dossett et
al. (2010) in samples from 26 black raspberry seedling populations, but is 2.5-3.0
times the amount of cyanidin-3-glucoside detected in the fruit of rr plants (Table 4.1).
Some excess cyanidin-3-glucoside may have been converted into cyanidin-3sambubioside by the addition of a xylose to the 2-position on the glucose. This
accounts for the substantial increase in the amount of cyanidin-3-sambubioside,
84
which normally comprises only 2-6% of the total anthocyanins (Dossett et al., 2010).
In the normal sample from this study, cyanidin-3-sambubioside accounted for 6% of
the total anthocyanins, while 1.5 to nearly 4 times the amount of cyanidin-3sambubioside was present in the rr fruit, accounting for up to 73% of the total
anthocyanins (Table 4.1). This result suggests that in the absence of cyanidin-3rutinoside as a substrate for xylosyltransferase (XyT), xylose is added to an increased
amount of cyanidin-3-glucoside resulting in higher levels of cyanidin-3sambubioside. Alternatively, cyanidin-3-sambubioside may be a substrate from
which cyanidin-3-xylosylrutinoside is made and the accumulation of cyanidin-3sambubioside is because it is no longer being used for this. While both of these
routes of synthesis are illustrated in Figure 4.2, further work examining the kinetics
and substrate specificities of these enzymes is needed to determine which is
responsible for production of cyanidin-3-xylosylrutinoside.
The presence of pelargonidin-3-glucoside in rr fruit was not surprising as this
is a precursor for pelargonidin-3-rutinoside, which is normally observed (Kamsteeg
et al., 1979). The identity of pelargonidin-3-glucoside was confirmed by comparison
of peak retention times from pelargonidin-3-glucoside in strawberry (main
strawberry anthocyanin; Hong and Wrolstad, 1990b) as well as by ESI-MS/MS (Fig.
1). Trace amounts of pelargonidin-3-glucoside were reported in black raspberry fruit
by Wu et al. (2006), however, other studies (Dossett et al., 2008; Tian et al., 2006a,
2006b; Tulio, et al., 2008; Wyzgoski et al., 2010) have not detected pelargonidin-3glucoside in black raspberry, suggesting that most of it is normally converted to
85
pelargonidin-3-rutinoside. Pelargonidin-3-glucoside levels were only slightly lower
than the amount of pelargonidin-3-rutinoside that would otherwise be expected. In
the normal sample from Lewis and Clark State Park, 11.8 mg·100 mL-1
pelargonidin-3-rutinoside was detected, accounting for roughly 3% or the total
anthocyanins. Dossett et al. (2010) found between 4.2 and 14.5 mg·100 mL-1
pelargonidin-3-rutinoside in seedlings from black raspberry crosses. The sample of
rr fruit with the highest pelargonidin-3-glucoside contained only 4.4 mg·100 mL-1.
Pelargonidin-3-glucoside comprised 3-5% of the total anthocyanins in these samples,
a similar proportion to what would be expected, however the total amount was far
less.
The data also allow us to infer the probable pathway for production of trace
amounts of peonidin-3-rutinoside in black raspberry fruit. Peonidin is produced by
methyltransferase (MT) activity on the 3’ position of the cyanidin B-ring. Jonsson et
al. (1983) showed that MT was the final step in the production of peonidin-3-(pcoumaroyl)-rutinoside-5-glucoside
from
cyanidin-3-(p-coumaroyl)-rutinoside-5-
glucoside in P. × hybrida. The presence of peonidin-3-rutinoside in the two samples
(reps 3 and 4) containing all or some normally pigmented fruit from ORUS 4141 is
in contrast to the absence of peonidin-3-rutinoside in rr fruit from that population
(rep 2) and all three rr samples from ORUS 4143. The absence of peonidin-3glucoside in these samples as a precursor to peonidin-3-rutinoside synthesis suggests
that peonidin-3-rutinoside is produced as a result of MT activity on cyanidin-3rutinoside (Fig. 4.2).
86
While fruit of the normal black raspberry sample contained 423.9 mg·100
mL-1 of total anthocyanins, fruit from the rr plants ranged from 77.5 to 134.4 mg·100
mL-1. This is far less than the range of 244.8-541.3 mg·100 mL-1 found by Dossett et
al. (2010) and is also less than the rest of the > 400 samples from other wild
populations harvested in 2010 (data not shown). This reduction in total anthocyanins
supports the observation that cyanidin-3-glucoside did not accumulate in proportion
to the reduction in cyanidin-3-rutinoside and its derivatives in the 3RT mutants. It
also indicates that there may be some feedback mechanism by which cyanidin-3glucoside was no longer produced or was produced at a lower rate once it began to
accumulate in the fruit. This could be because glucosyltransferase (GT) activity
reaches equilibrium in raspberry fruit at a relatively low concentration of cyanidin-3glucoside, or because of feedback to some limiting step at an earlier part of the
pathway. Either way, it seems that in black raspberry fruit, it may be important for
cyanidin-3-glucoside to be anabolized for synthesis of other anthocyanins if high
total anthocyanin content is desired. If this is the case, selecting alleles for more
efficient GT, RT, and XyT involved in anthocyanin biosynthesis, or for plants that
produce more types of anthocyanins, may be a good strategy for breeding for high
total anthocyanins. Alternatively, a decrease in cyanidin-3-glucoside and other
anthocyanins may result in higher production of other phenolics that may be of
interest to nutraceutical or other markets for fruit that don’t require higher
anthocyanin content but may be focused on the concentration of other compounds.
87
In addition to gaining insight regarding the biology of anthocyanin
modifications in black raspberry, studying fruit from these 3RT mutants may also
give researchers the opportunity to study the impact of raspberry anthocyanin
composition on color of whole fruit and processed products. Wiering and de
Vlaming (1984) indicated that anthocyanidin-3-rutinosides result in a bluer color in
P. × hybrida flowers while flowers containing only anthocyanidin-3-glucosides
appear redder. If this is also the case in black raspberry, it may be a reason for some
of the color difference between black and red raspberry fruit besides differences in
anthocyanin concentration. Stintzing et al. (2002) found that the addition of xylose to
cyanidin-glycosides lowered the visual detectability threshold of anthocyanins.
Stintzing et al. (2002) also noted that the color contribution of most anthocyanins
was less than the percentage of their HPLC peak area. Cyanidin-3-glucoside
accounted for 83% of the anthocyanins in fruit of R. laciniatus Willd., but only
accounted for about 50% of the color. Fruit pH, as well as compositional and/or
physical factors may also play a role in perceived color of whole fruit and processed
products such as juice. While fruit of rr plants were not noted for unusual appearance
at the time of harvest, their lighter color, due presumably to lower anthocyanin
concentration, was noted during the preparation of juice samples.
88
Conclusions
Anthocyanidin-3-rutinosides and their derivatives account for as much as
90% of black raspberry anthocyanins (Dossett et al., 2010; Tian et al., 2006a; Tulio,
et al., 2008; Wyzgoski et al., 2010). The identification of a black raspberry 3RT
mutant offers an opportunity to identify a major gene responsible for anthocyanin
modification in black raspberry as well as other alleles with altered 3RT efficiency,
affecting not only the amount of total anthocyanins but their proportions as well, and
leading to a better understanding of raspberry anthocyanin synthesis and its affects
on color and quality. Small quantities of seed of ORUS 4141 (PI 653314) and ORUS
4143 (PI 653316) are available by request from the USDA-ARS National Clonal
Germplasm Repository in Corvallis, OR.
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Table 4.1. Anthocyanin profiles and total anthocyanins by HPLC for juice from seedlings of wild black raspberry populations
from Lewis and Clark State Park (ORUS 4141, Yankton, SD, USA) and Union Grove State Park (ORUS 4143, Beresford,
SD, USA) and grown in Corvallis, OR, USA. All units are in mg·100 mL-1. Values shown represent fruit bulked by plot (rep,
replication). Proportions of the individual anthocyanins are included (in %). Due to disease, plants from plots ORUS 4141
rep 1 and ORUS 4143 rep 4 did not produce sufficient fruit for analysis. Identification was performed by HPLC/DAD/ESIMS/MS.
Population
Rep
cyanidin-3sambubioside
ORUS 4141
ORUS 4141
ORUS 4141
ORUS 4143
ORUS 4143
ORUS 4143
2
3
4
1
2
3
45.6
23.9
22.2
90.7
33.0
92.9
56%
6%
15%
67%
43%
73%
cyanidin-3glucoside /
cyanidin-3xylosylrutinoside
33.0z 41%
282.2y 67%
61.2y 42%
39.4 z 29%
40.8 z 53%
30.9 z 24%
cyanidin-3rutinoside
npx
103.5 24%
58.1 39%
np
np
np
pelargonidin3-glucoside
3%
ndw
2.3 2%
4.4 3%
3.7 5%
3.7 3%
pelargonidin
-3-rutinoside
2.8
peonidin-3rutinoside
np
11.8
3.5
np
3%
2%
np
np
np
2.5
1.0
1%
0.7%
np
np
np
Total
anthocyanins by
HPLC/DAD
81.3
423.9
147.2
134.4
77.5
127.6
z
In ORUS 4141 rep 2 and in ORUS 4143 (all reps), cyanidin-3-glucoside only. No cyanidin-3-xylosylrutinoside was
detected.
y
cyanidin-3-xylosylrutinoside is the major peak with coelution with a minor amount of cyanidin-3-glucoside.
x
not present.
w
not detected.
92
93
2a & b
120
0
100
0
80
0
60
0
40
0
20
0
0
3
1
5
6
8
Absorbance at 520 nm
(mAU)
30
0
25
0
ORUS 4141, rep 3
10
2a & b
1
3 2
1
4
1
6
6
18
20
0
2
0
2
2
2
4
ORUS 4141, rep 4
15
0
1
10
0
5
0
4
5
6
0
6
8
50
0
1
1
0
1
2
1
4
1
6
1
8
2
0
2
2
24
40
0
30
0
ORUS 4143, rep 1
2a
20
0
10
0
4
0
6
8
1
0
1
2
1
4
1
6
1
8
2
0
2
2
Time
(min)
2
4
Fig. 4.1. Chromatograms showing anthocyanin profiles of black raspberry juice from
normal (ORUS 4141, rep 3), mixed (ORUS 4141, rep 4), and mutant (ORUS 4143,
rep 1; rr) samples from seedlings grown in Corvallis, OR, USA and harvested in
2010. Peak numbers are identified as follows [m/z of molecular ions and their
fragments]: 1) cyanidin-3-sambubioside (581, 287); 2) a, cyanidin-3-glucoside (449,
287; minor) and b, cyanidin-3-xylosylrutinoside (727, 581, 287); 3) cyanidin-3rutinoside (595, 287); 4) pelargonidin-3-glucoside (433, 271); 5) pelargonidin-3rutinoside (580, 271); and 6) peonidin-3-rutinoside (609, 301).
94
Fig. 4.2. Pathway for anthocyanin modifications in black raspberry. Genes (italicized)
follow the designations by Barritt and Torre (1975b) and Jennings and Carmichael
(1980) while enzymes (XyT = xylosyltransferase, 3RT = UDP-rhamnose
anthocyanidin-3-glucoside rhamnosyltransferase, MT = methyltransferase) are in
regular type. Hypothetical steps are illustrated by dashed arrows. Designation of
sugars attached to aglycones is as follows: Glc = Glucose, Rha = rhamnose, and Xyl
= xylose.
95
Chapter 5:
Genetic Diversity in Wild and Cultivated Black Raspberry
Evaluated by Simple Sequence Repeat Markers
Michael Dossett, Nahla V. Bassil, Kim S. Lewers, Chad E. Finn
96
Abstract
Breeding progress in black raspberry (Rubus occidentalis L.) has been limited
by a lack of genetic diversity in elite germplasm. Black raspberry cultivars have been
noted for showing very few phenotypic differences and seedlings from crosses
between cultivars for a lack of segregation for important traits. Despite these
challenges, little molecular work has been done to explore genetic diversity and
relationships in wild and cultivated black raspberry germplasm. Microsatellite, or
simple sequence repeat (SSR), markers are highly polymorphic codominant markers
useful for studying genetic diversity, population genetics, genetic fingerprinting and
other applications. Using 21 polymorphic SSR markers, we examined genetic
diversity in 148 wild and cultivated black raspberry accessions. Black raspberry
cultivars clustered tightly and showed higher than expected heterozygosity while
heterozygosity of wild accessions was low. Relationships between wild black
raspberry accessions were poorly resolved and regional clusters were mostly absent
from our analysis. Our results indicate that wild black raspberry germplasm is a
relatively untapped resource available for future breeding.
Introduction
The black raspberry, often called “blackcap”, was first domesticated in the
1830s (Hedrick, 1925). A member of the Rosaceae, it is diploid (2n=2x=14) and
97
belongs to the same subgenus (Idaeobatus) as the red raspberry (R. idaeus L. and R.
strigosus Michx.), with which it readily crosses. Native to eastern North America
from New Brunswick to the Carolinas and as far west as Kansas and western
Nebraska, black raspberries are typically found in disturbed habitats and near forest
edges (Jennings, 1988). West of the Rockies, it is superseded by R. leucodermis
Dougl. ex Torr. & Gray (Hitchcock and Cronquist, 1973), which occupies a similar
niche and is similar in appearance but with more coarsely toothed leaves, spinier
canes, and softer, purplish fruit. Both species are somewhat unusual among diploid
members of the Idaeobatus for their self-compatible flowers (Jennings, 1988).
The black raspberry industry in North America has undergone a slow but
steady contraction since the 1920s due in large part to disease and a lack of adapted,
disease-resistant cultivars. Today, growers in Oregon, the leading production region,
typically see a decline in production after the second harvest and remove fields after
only three or four seasons because of decreased profitability (Halgren et al., 2007).
At the same time, demand for black raspberry fruit has increased in recent years in
large part because of studies outlining the potential health benefits of black raspberry
consumption (Kresty et al., 2001; Seeram et al., 2006; Seeram, 2008; Stoner et al.,
2005, 2008). These factors have combined to create a renewed interest in breeding
better cultivars that meet the demands of growers and consumers.
Historically, progress in breeding black raspberry has been limited by a lack
of variation and segregation for important traits in elite germplasm. This has long
been recognized and attempts to broaden the genetic base of black raspberry
98
breeding populations by using other Rubus species date back to the 1950s (Drain,
1956; Slate and Klein, 1952; Williams, 1950). The lack of genetic diversity is so
acute that Ourecky (1975) felt that no future progress would be made in breeding
black raspberry without the use of other species. However, in contrast to red
raspberry, in which interspecific hybridization has played a major role in the
introgression of new traits of interest, this approach has been of limited success in
black raspberry. ‘Earlysweet’, released in 1996, is the first, and only, black raspberry
cultivar reported to have another species, R. leucodermis, in its background (Galletta
et al., 1998).
Only a few recent studies have attempted to quantify the genetic variation
present in black raspberry germplasm. Weber (2003) examined genetic diversity in
14 cultivars and two wild selections from New York using random amplified
polymorphic DNA (RAPD) markers. Genetic diversity was quite low; on average,
there was 81% similarity among polymorphic markers, however, more than half of
this variability was accounted for by ‘Black Hawk’, ‘Cumberland’, ‘John Robertson’,
and the two wild selections. The remaining 11 genotypes had a collective marker
similarity of 92%. Weber (2003) asserted that many cultivars that originated as
chance seedlings were probably from open pollination of other cultivars. While this
work yielded valuable information about the apparent lack of variability and
relationships between black raspberry cultivars, RAPD markers lack the
reproducibility desired for genetic fingerprinting and large scale population studies.
Nybom and Schaal (1990) used restriction fragment length polymorphism (RFLP)
99
markers to document genetic diversity in a wild black raspberry population in
Missouri. They found 15 unique genotypes among 20 plants sampled along a 600 m
stretch of roadside, and suggested that the main mode of plant recruitment in this
population was through sexually produced seed leading to intrapopulation genetic
diversity.
Simple sequence repeat (SSR) or microsatellite markers are robust, highly
polymorphic, codominant markers giving them a distinct advantage over RAPD and
RFLP markers for applications in population genetics, genetic diversity studies, and
DNA fingerprinting. Microsatellite markers have been developed from expressed
sequence tag (EST) and genomic libraries in red raspberry (Castillo, 2006; Graham
et al., 2004) and blackberry (Rubus L. subgenus Rubus) (Amsellem et al., 2001;
Castillo et al., 2010; Lewers et al., 2008; Lopes et al., 2006). More recently, work
has also started to develop SSRs from black raspberry ESTs (unpublished data).
Using SSR markers, Dossett et al. (2010) found 12 black raspberry cultivars to be
more closely related to each other than to any of the four wild accessions examined.
These results, along with those of Weber (2003) and Nybom and Schaal (1990),
suggest that there may be a great deal more genetic diversity in wild populations than
in current cultivars.
Surprisingly, beyond a few selections made in the late 19th and early 20th
centuries, there is little record of the use of wild R. occidentalis as a source of
genetic diversity for breeding improved black raspberry cultivars, and no record of
any effort to systematically collect and evaluate germplasm from across the entire
100
range of the species. Dossett et al. (2008) found increased vigor and adaptability in
progeny of a wild black raspberry selection from North Carolina. Dossett and Finn
(2010) found aphid resistance in wild black raspberry germplasm, a trait that will be
of great benefit in developing new virus resistant cultivars. It appears that wild black
raspberry germplasm could be beneficial in developing better adapted and more
disease resistant cultivars. In this study, we investigate the level of genetic variation
present in wild populations, and elite germplasm.
Materials and Methods
Plant Materials. During the summer of 2006, friends and colleagues in eastern North
America, within the native distribution of R. occidentalis, were solicited to send seed
or fruit from wild plants in their area. Additional seed was obtained in 2007 through
a similar request and from collecting trips across the southern and western edges of
the native range (Hall et al., 2009; Hummer et al., 2008a, 2008b). Through these
efforts, seeds were obtained from more than 150 locations across the range,
including 27 states and two Canadian provinces. Upon arrival in the lab, seeds were
extracted from the fruit, dried, and stored in a cool dry place until scarification.
Additional seed was obtained from R. occidentalis seed lots held at the National
Clonal Germplasm Repository (NCGR) in Corvallis, OR. Seeds were treated to
promote germination using the methods of Dossett and Finn (2010), and a single
seedling from each population from which seed was successfully germinated was
101
randomly selected for inclusion in this study. In addition, each of the black raspberry
cultivars and wild accessions currently available as clones at the NCGR were
included in this study, for a total of 21 cultivars and 137 wild accessions (Table 5.1,
Fig. 5.1). Naturally occurring hybrids with red raspberry (as recognized by their
densely spined canes, and differences in leaf shape and leaflet number) were noted in
seedlings of a few populations and were deliberately avoided when sampling
seedlings for this study. Plants showing morphology consistent with polyploidy
(primarily leaf shape and appearance of leaf venation, see Hull and Britton, 1956)
were identified in two of the populations and these seedlings were also excluded
from sampling for this study. Two wild seedlings of R. leucodermis, one from
Washington, the other from Oregon, of R. leucodermis were included for comparison
and dendrogram construction, but were not included in measurements of allelic
diversity.
DNA extraction and amplification. DNA was extracted from freshly growing young
leaf tissue with the Gentra Puregene kit (Qiagen, Valencia, CA) using the optional
RNAse A treatment.
Rubus SSR primer sequences were selected from published reports in red
raspberry (Graham et al., 2004) and blackberry (Castillo, 2006; Lopes et al., 2006;
Lewers et al., 2008). Dossett et al. (2010) described the transferability of many of
these Rubus SSR primers to black raspberry. These primers, and two previously
unreported black raspberry EST SSR primer pairs, are summarized in Table 5.2.
102
Optimum annealing temperatures was determined by gradient polymerase chain
reaction (PCR) from 50 °C to 65 °C in ‘Munger’ using non-fluorescent primers.
After an initial denaturation at 94 °C for 3 min, DNA was amplified for 35 cycles in
a PTC-225 gradient thermal cycler (Bio-Rad, Hercules, CA) programmed for a 40 s
denaturation step at 94 °C, a 40 s annealing step at the optimum annealing
temperature of the primer pair and a 40 s extension step at 72 °C. A final extension
step at 72 °C for 30 min was included. Non-fluorescent PCR reactions were
performed in a volume of 10 µl and bands visualized by ethidium bromide staining
after separation by 2% agarose gel electrophoresis. PCR was then performed on all
samples with fluorescently labeled (WellRed D2, D3, or D4) forward primers at the
appropriate annealing temperature in a volume of 15 µl. For some SSRs, instead of
fluorescently
labeling
all
forward
primers,
the
M13
sequence
TGTAAAACGACGGCCAGT was added to the 5’ end of the forward primer (Table
2) and a fluorescently labeled (WellRed D2, D3, or D4; Integrated DNA
Technologies, Inc. Coralville, Iowa) M13 primer was used in the PCR, following the
procedure outlined by Schuelke et al. (2000). Fluorescently labeled PCR products
were separated by capillary electrophoresis using a Beckman CEQ 8000 genetic
analyzer (Beckman Coulter, Fullerton, California) for all samples. The reverse
primer for Rub1C6 was pigtailed (Brownstein et al., 1996) to minimize the
occurrence of split peaks and the difficulties encountered in fragment analysis
following capillary electrophoresis.
103
Data analysis. The data were compiled and analyzed with PowerMarker (Liu and
Muse, 2005). Expected and observed heterozygosity (He, Ho, Nei, 1987) and
polymorphism information content (PIC, Botstein et al., 1980; Liu, 1998) were
estimated for all black raspberry genotypes together, as well as separately for
cultivated and wild genotypes. A dendrogram (Fig. 5.2) was constructed based on the
proportion of shared alleles distance measure (Bowcock et al., 1994) using
unweighted pair-group method analysis (UPGMA). A separate neighbor-joining (NJ)
dendrogram was constructed from a cluster within the UPGMA tree comprised of
most black raspberry cultivars and a few wild black raspberry accessions (Fig. 5.3).
The bootstrap option of PowerMarker was used to create 1000 dendrograms and
MEGA version 4 software (Tamura et al., 2007) was used to generate and edit a
consensus dendrogram. Principal component analysis (PCA) was performed using a
similarity matrix based on Euclidean distances with NTSYS-pc (version 2.1; Exeter
Software, Setauket, NY).
Results and Discussion
Twenty-one SSR primer pairs amplified one or two alleles in each of the 21
cultivated and 125 wild R. occidentalis accessions. In 12 additional wild accessions,
more than two alleles were amplified by one or more of the primer pairs studied.
This may be the result of introgression of alleles from red raspberry or other Rubus
species, duplication of some genome regions, or polyploidy. Individuals amplifying
104
more than two alleles for any primer pair (ORUS 3779, ORUS 3789, ORUS 3795,
ORUS 3803, ORUS 3823, ORUS 3827, ORUS 3910, ORUS 4111, ORUS 4122,
ORUS 4141, ORUS 4142, and ORUS 4147) were excluded from the analysis and the
remaining data were treated as though each SSR primer pair amplified a single locus.
Allelic diversity among the 21 black raspberry cultivars was very low, with
three or fewer alleles present at 15 of 21 loci (Table 5.3). The 21 SSR loci were
unable to distinguish between six of the cultivars: Bristol, Cumberland, Munger,
New Logan, Plum Farmer, and Shuttleworth (Fig. 5.3). This is in contrast to previous
work using 19 of the same SSRs (Dossett et al., 2010) that found differences
between some of these cultivars. During the course of this study, we found that
differences in primer stocks led to some fragments having been incorrectly sized in a
subset of the data from Dossett et al. (2010). Once this problem was discovered,
PCRs for samples at the suspect loci were repeated and the correct alleles were
verified. The most recently named cultivar of this group (Bristol) was released nearly
80 years ago, and it is possible that mislabeling of plants at some point in the past led
to this result. While Hedrick (1925) considered several of these clones to be distinct,
Ourecky (1975) noted difficulty in distinguishing between black raspberry cultivars
as well as a lack of segregation for important traits in black raspberry seedlings. This
may have been due in part to identical clones being evaluated under different names.
Alternatively, these genotypes may be distinct but cannot be distinguished with
existing SSR markers. Using RAPD markers, Weber (2003) was able to distinguish
between each of the 14 black raspberry cultivars examined. In that study, ‘Bristol’,
105
‘Munger’, ‘New Logan’, and ‘Plum Farmer’ had very high marker similarity
(average = 97%); ‘Cumberland’ was somewhat less similar (average 86% similarity);
and ‘Shuttleworth’ was not included. It is unlikely that the differences observed by
Weber (2003) are due solely to the lack of reproducibility of RAPD markers as
previously reported (Büscher et al., 1993; Jones et al., 1997; MacPherson et al.,
1993). In either case, our data highlights the need for better genomic resources and
markers to reliably distinguish between closely related black raspberry genotypes, as
well as a need for greater genetic diversity in material used in breeding. Further
study will be needed to determine whether there are real performance differences
between these five clones in the field. Clones from alternate sources should also be
fingerprinted. Unfortunately, ‘New Logan’, ‘Plum Farmer’, and ‘Shuttleworth’ are
no longer widely available and may be among the many black raspberry cultivars
that have been lost over the last 100 years.
Based on the similarity of their alleles, the majority of black raspberry
cultivars clustered tightly in one relatively well defined group in the UPGMA
dendrogram (Fig. 5.2). The average branch length (distance of shared alleles)
separating all of the black raspberry cultivars was 0.26. A neighbor-joining
dendrogram depicting the genotypes within this group shows good bootstrap support
for several of the pairings (Fig. 5.3). ‘Explorer’, the one cultivar falling outside of
this group, was selected from crosses of wild plants from New York and Arkansas
for its unusual fall-fruiting habit (Tallman, 2007) and was therefore not expected to
show a close relationship to other cultivars. Within the cluster of 20 cultivars (Fig.
106
5.3), there were also nine wild black raspberry accessions (ORUS 3801, ORUS 3816,
ORUS 3824, ORUS 3844, ORUS 3857, ORUS 3931, ORUS 3956, ORUS 4110, and
ORUS 4130), some of which consistently paired with cultivars (e.g. ORUS 3956
with ‘Jewel’). With the exception of ORUS 3931, which was noted in the field for its
distinct morphology, each of these had larger than average fruit and/or came from
seed lots that segregated for plants lacking the normal waxy, glaucous bloom on their
canes (data not shown). Dossett (2007) noted segregation for non-glaucous canes in
progeny of some black raspberry cultivars, and the presence of one or both of these
traits in these populations suggests that they are likely derived from escaped
cultivated germplasm.
Despite the low allelic diversity found among black raspberry cultivars, novel
alleles not found in the wild genotypes were present at three loci (Table 5.3). Further
examination revealed discrepancies between SSR fingerprint and the stated pedigrees
of some cultivars. The published pedigree of ‘Jewel’ is (‘Bristol’ × ‘Dundee’) ×
‘Dundee’. However, in our study, ‘Jewel’ had alleles at multiple SSR loci that are
not carried by either ‘Bristol’ or ‘Dundee’ (as illustrated by 112 at ssrRhcBA23 and
169 at Rubus110a, Table 5.4). This indicates that either the published pedigree is
incorrect, or the identity of the ‘Jewel’, ‘Dundee’, or ‘Bristol’ used in this study is
incorrect (Table 5.4). ‘Jewel’ and ‘Huron’ (‘Rachel’ × ‘Dundee’) were the only two
individuals sharing a 112 bp allele at ssrRhCBA23, the most polymorphic locus in
this study (Table 5.4), suggesting that either ‘Huron’ or ‘Rachel’ may be an ancestor
of ‘Jewel’. The identity of ‘Huron’ in this study also does not match its reported
107
pedigree as it does not share an allele with ‘Dundee’ at Rubus 275a (Table 5.4).
‘Allen’ (‘Bristol’ × ‘Cumberland’) and ‘Haut’ [(‘Cumberland’ selfed × selfed) ×
‘Bristol’ selfed] also have alleles that cannot be traced to either of their reported
parents (as shown by 158 at Rubus126b for ‘Allen’ and 128 at Rubus275a and 187 at
Rubus110a for ‘Haut’, Table 4). ‘Haut’ and ‘Huron’ were the only two cultivars
sharing a 128 bp allele for Rubus 275a (Table 5.4).
Similarly, ‘Earlysweet’ is reported to have R. leucodermis as one of its
grandparents (Galletta et al., 1998). Alleles observed in the two R. leucodermis
accessions fell outside the size range of R. occidentalis at seven loci (Table 5.2) and
were unique to R. leucodermis at six other loci where there was size overlap (data
not shown). While only two R. leucodermis genotypes were available in this study
for comparison, SSR alleles found in ‘Earlysweet’ were characteristic of other R.
occidentalis cultivars at every locus, and alleles in the size range of R. leucodermis
were not observed. This, combined with its close clustering within the group of other
cultivars, suggests that ‘Earlysweet’ is may not be one quarter R. leucodermis as
reported. ‘Earlysweet’ [(‘Haut’ × R. leucodermis) × open-pollinated] may have
instead originated from contamination of the pollen used in the cross, or from
contamination of the open-pollinated seed lot. In this study, ‘Earlysweet’ grouped
closely with ‘Ebonee’ (‘Cumberland’ open-pollinated), possibly due to shared alleles
from ‘Cumberland’, a common ancestor. ‘Earlysweet’ and ‘Dundee’ were the only
two individuals in the study with a 188 bp allele at Rubus 123a, indicating that
‘Dundee’ may be a parent of ‘Earlysweet’ (Table 5.4). This close relationship is also
108
supported by RAPD markers (Weber, 2003). Similarly, ‘Mac Black’ and ‘Black
Knight’ were the only two individuals that shared a 209 bp allele at Rubus 262b
(Table 5.4). While the pedigree of ‘Mac Black’ is unknown, ‘Black Knight’
(‘Johnson Everbearing’ selfed) predates ‘Mac Black’ by about 20 years and may be
in its lineage. Because of its erect growth habit and very late fruit maturity, as
compared to other black raspberry cultivars, there has been speculation that ‘Mac
Black’ may have R. idaeus in its ancestry (Makielski, personal communication).
However, SSR alleles in ‘Mac Black’ were characteristic of R. occidentalis,
matching those found in other cultivars at every locus. This, along with its clustering
with the other black raspberry cultivars, casts some doubt on this hypothesis.
SSR analysis also revealed a surprising level of heterozygosity in black
raspberry cultivars. At every polymorphic SSR locus examined, observed
heterozygosity in the cultivars was higher than expected heterozygosity. This
shouldn’t be a big surprise since the process of selection and breeding, particularly in
a clonally propagated crop such as black raspberry, can lead to highly heterozygous
breeding populations. What is slightly surprising, however, is that this heterozygosity
has been maintained despite some inbreeding in many cultivars that should lead to a
loss of heterozygosity. While pedigree information is missing or sparse for many
cultivars, several are known to be parents and/or grandparents of others. This is
indicative of inadvertent selection for heterozygosity in the process of selecting for
the best performers, and leads one to suspect that homozygosity may lead to
inbreeding depression in black raspberry despite “conventional wisdom” that black
109
raspberries do not suffer from inbreeding depression (Haskell, 1960; Ourecky, 1975).
Dossett (2007) and Dossett et al. (2008) noted that progeny of a wild black raspberry
selection from North Carolina, NC 84-10-3, when crossed to cultivars, outperformed
and had higher vigor than progeny of crosses among cultivars. This is despite
observations that NC 84-10-3 had very low vigor and never grew to be large in the
field (Dossett 2007).
In this study, NC 84-10-3 was heterozygous at only one of the 21 loci
examined (data not shown), suggesting a degree of inbreeding. In fact, the wild
genotypes in this study had lower than expected heterozygosity at every polymorphic
SSR locus (Table 5.3). This is not entirely unexpected; subdivision of wild black
raspberry populations will lead to an apparent deficiency of heterozygotes and the
sampling method violates Hardy-Weinberg expectations. Despite this, the rate of
observed heterozygosity (0.21) is less than half that observed in the cultivars (Table
5.3). The reasons for this are unclear, but may be due to bottlenecking and/or
isolation of wild populations from one another. Further sampling from within these
populations is needed to better understand the reasons for the observed
homozygosity before firm conclusions can be made about the causes.
With the high degree of homozygosity in mind, one should consider the
possibility that inbreeding depression may be a limiting factor in the field
performance of some of these seedlings and their value in breeding may only become
evident from the performance of their progeny from crosses with unrelated
110
germplasm. At the same time, further study should be undertaken to examine the
impact of inbreeding on black raspberry performance.
In contrast to black raspberry cultivars, wild black raspberry germplasm
appears to be relatively diverse. In general, branch lengths separating the wild
genotypes are longer than those separating the cultivars and bootstrap support for
groups of wild accessions was poor, indicating that these accessions are more
distantly related to each other and that their relationships were not well resolved. The
average branch length (distance of shared alleles) separating wild black raspberry
accessions in this study was 0.53, more than twice that of the cultivars. UPGMA
clustering (Fig. 5.2) illustrates a general lack of grouping based on geographical
location. For example, wild plants from Indiana (ORUS 3794) and North Carolina
(NC 84-10-3) grouped together as did plants from New Brunswick (ORUS 3777)
and South Carolina (ORUS 4114). However, a few groups of accessions from
neighboring locations were scattered throughout the dendrogram. There is also one
large group which, with the exception of single samples from Kentucky, Minnesota,
New York, and Maryland, consists entirely of accessions from Tennessee, Georgia,
and North Carolina. The lack of resolution in relationships among wild black
raspberry populations, combined with the general lack of strong regional clusters,
may be an indication that black raspberry diversity has not been exhaustively
sampled. This also suggests that black raspberry populations may be welldifferentiated from each other but not in a strongly geographical manner. Future
work investigating the extent of diversity and relationships within and between many
111
of these wild populations should provide insight into the degree of differentiation
between wild populations and answer questions regarding whether certain areas of
the range contain more allelic diversity than others. This information would be useful
for future efforts to collect and preserve genetic diversity in wild black raspberry
germplasm.
Principal component analysis did not provide better resolution of the data but
did support some of the clusters already observed in the dendrogram. The first three
eigenvalues collectively explain only 9.6% of the variance. The first, however,
separated black raspberry cultivars from the majority of the wild germplasm (Fig.
5.4). Wild accessions that clustered with the cultivars in Fig. 5.2 also grouped with
the cultivars on the positive side of this axis. ‘Explorer’ and the wild accessions that
clustered with it in the UPGMA dendrogram also fell near these on the positive end
of the first axis. A few additional wild accessions fell in this area, including ORUS
3799, ORUS 3819, ORUS 3851, ORUS 3947, ORUS 3955, and ORUS 4124. ORUS
4124 has been noted in field evaluations for fruit weight and seed lots of ORUS 3799
and ORUS 4124 segregate for non-glaucous canes (data not shown) that may be an
indication of cultivated ancestry. The second PCA axis provides some separation
between the rest of the black raspberry cultivars and the UPGMA cluster that
includes ‘Explorer’ and five wild accessions (ORUS 3811, ORUS 3830, ORUS 3833,
ORUS 3856, and ORUS 4134). These were located towards the negative end of axis
2 with the rest of the cultivars spread out along this axis. Otherwise, separation of
groups along the second and third axes was relatively poor and groups of wild
112
accessions were not well resolved. Eigenvalues four and five (data not shown) each
explain only about 2% of the variance, and plotting these does not help further
resolve relationships in black raspberry germplasm.
Conclusions
Overall, it appears that the vast majority of genetic diversity available in R.
occidentalis remains untapped in the development of new cultivars. While several
cultivars that have not been lost over the last 100 years are reputed to have originated
as wild seedlings that were discovered and brought into cultivation because of their
superior horticultural traits (Hedrick, 1925), it is now clear that the remaining black
raspberry cultivars are very closely related to each other and many of the “wild”
selections named as cultivars were probably seedlings of cultivated plants. The few
apparently wild accessions that clustered with cultivars in this study have traits such
as larger than average fruit, suggesting that they may be the offspring of cultivated
plants. Conversely, this also shows that characterization of wild-collected black
raspberry germplasm with SSR markers may be a useful tool in the future for
identifying whether wild plants with good horticultural attributes are truly wild or
closely related to cultivated material.
Even the most recently developed black raspberry cultivars are not more than
a few generations removed from truly wild ancestors. This knowledge, combined
with the apparent diversity among wild plants available today, suggests that
113
significant progress in breeding improved cultivars may be possible from a few
generations of crossing and selection from between these wild populations without
requiring further use of cultivated black raspberry germplasm. The use of this wild
germplasm combined with existing cultivars should lead to even faster gains for
some commercially important traits, such as fruit weight.
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117
Table 5.1. U.S. Department of Agriculture- Agricultural Research Service plant
introduction (PI) number, accession name, origin, and type, for 137 wild and 21
cultivated black raspberry (Rubus occidentalis L.) accessions studied.
PI no.
Name
Origin
Type
653296
653327
652978
652975
652976
653294
653298
652977
653328
NAz
653329
553949
553949
553950
653331
653335
653332
653330
653333
NA
653334
653336
652984
653299
653303
653301
651846
653302
653300
651848
653337
653338
653343
653344
NA
653341
ORUS 4123
ORUS 3779
HDF-039
ORUS 4117
ORUS 4119
ORUS 4120
ORUS 4122
ORUS 4121
ORUS 3780
ORUS 3789
ORUS 3781
ORUS 3946
CRUB 641.002
CRUB 642.001
ORUS 3796
ORUS 3800
ORUS 3797
ORUS 3794
ORUS 3798
ORUS 3795
ORUS 3799
ORUS 3801
ORUS 4126
ORUS 4124
ORUS 4129
ORUS 4127
ORUS 4130
ORUS 4128
ORUS 4125
ORUS 3802
ORUS 3803
ORUS 3804
ORUS 3811
ORUS 3812
ORUS 3809
ORUS 3808
Mentone, AL
Litchfield County, CT
Appalachian Trail, GA
Clayton, GA
Clayton, GA
Clayton, GA
Dahlonega, GA
Union County, GA
Story County, IA
Arenzeville, IL
Iroquois County, IL
Waukegan County, IL
Waukegan County, IL
Waukegan County, IL
Greene County, IN
Greene County, IN
Hendricks County, IN
Putnam County, IN
Sullivan County, IN
Vigo County, IN
Vigo County, IN
southern IN
Alma, KS
Bonner Springs, KS
Fort Riley, KS
Manhattan, KS
Minneapolis, KS
Ogden, KS
Perry Lake, KS
Fayette County, KY
Berkshire County, MA
Berkshire County, MA
Allegany County, MD
Anne Arundel County, MD
Dorchester County, MD
Harford County, MD
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
118
Table 5.1. (continued)
PI no.
653342
NA
653339
653340
653350
653349
653347
653348
651849
653345
653346
NA
NA
553765
553766
NA
553764
NA
653323
653321
651847
653351
651851
653354
653324
651850
653353
NA
651852
653356
653357
653358
653359
553755
653311
653310
653308
618482
Name
ORUS 3810
ORUS 3806
ORUS 3805
ORUS 3807
ORUS 3821
ORUS 3820
ORUS 3817
ORUS 3819
ORUS 3815
ORUS 3814
ORUS 3816
ORUS 4109
ORUS 4110
ORUS 3948
ORUS 3949
ORUS 4111
ORUS 3947
ORUS 4112
ORUS 4149
ORUS 4148
ORUS 4147
ORUS 3823
ORUS 3827
ORUS 3828
ORUS 4150
ORUS 3824
ORUS 3826
ORUS 3833
ORUS 3830
ORUS 3832
ORUS 3835
ORUS 3837
ORUS 3838
NC 84-10-3
ORUS 4139
ORUS 4138
ORUS 4136
CRUB 1732.001
Origin
Howard County, MD
Howard County, MD
Washington County, MD
Washington County, MD
Camden, ME
East Vassalboro, ME
Gardiner, ME
Hallowell, ME
Monmouth, ME
Orono, ME
West Kennebunk, ME
Bath, MI
Benton Harbor, MI
Fred Russ State Forest, MI
Fred Russ State Forest, MI
Grand Ledge, MI
Oak Grove, MI
Okemos, MI
Belgrade, MN
Big Stone Lake National Wildlife Refuge, MN
Big Stone Lake State Park, MN
Cass County, MN
Dakota County, MN
Dakota County, MN
Hasty, MN
Ramsey County, MN
Ramsey County, MN
Cassville, MO
Fordland, MO
Fordland, MO
Madison County, NC
Rutherford County, NC
Rutherford County, NC
Zebulon, NC
Chadron, NE
Chadron State Park, NE
Halsey, NE
Nebraska City, NE
Type
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
119
Table 5.1. (continued)
PI no.
653305
653306
653309
653307
638243
638244
653363
653362
653360
653361
NA
618560
653364
653368
NA
NA
653372
653373
653369
653370
653371
NA
652971
652973
652974
653315
653318
653317
653319
652988
653316
653314
653389
653395
653374
653375
Name
ORUS 4133
ORUS 4134
ORUS 4137
ORUS 4135
ORUS 3955
ORUS 3956
ORUS 3843
ORUS 3842
ORUS 3839
ORUS 3840
ORUS 3841
ORUS 3951
ORUS 3844
ORUS 3849
ORUS 4107
ORUS 4108
ORUS 3854
ORUS 3856
ORUS 3851
ORUS 3852
ORUS 3853
ORUS 4185
ORUS 4113
ORUS 4114
ORUS 4115
ORUS 4142
ORUS 4145
ORUS 4144
ORUS 4146
ORUS 4140
ORUS 4143
ORUS 4141
ORUS 3904
ORUS 3915
ORUS 3857
ORUS 3863
Origin
North Loup State Recreation Area, NE
Pibel Lake State Recreation Area, NE
Valentine, NE
Victoria Springs State Recreation Area, NE
Manasquan Reservoir, NJ
Tom's River, NJ
Columbia County, NY
Dutchess County, NY
Ontario County, NY
Ontario County, NY
Ontario County, NY
Poughkeepsie, NY
Yates County, NY
Clermont County, OH
Hilliard , OH
Newton Falls, OH
Centre County, PA
Centre County, PA
Chester County, PA
Greene County, PA
Somerset County, PA
Charlestown, RI
Glassy Mountain, SC
Glassy Mountain, SC
Rich Mountain, SC
Clay County State Park, SD
East Sioux Falls, SD
Newton Hills State Park, SD
Oakwood Lakes State Park, SD
Pease Creek State Recreation Area, SD
Union Grove State Park, SD
Yankton, SD
Cannon County, TN
Cheatham County, TN
Davidson County, TN
Davidson County, TN
Type
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
120
Table 5.1. (continued)
PI no.
653376
618286
653377
653378
653379
653380
653381
653384
653385
653398
618287
653396
653397
653382
653390
653392
653393
653394
653383
653386
653387
653399
653401
653402
653400
653325
653326
553733
553734
553754
553735
553739
553770
657877
Name
ORUS 3864
NC 98-12-1
ORUS 3867
ORUS 3869
ORUS 3871
ORUS 3873
ORUS 3878
ORUS 3889
ORUS 3893
ORUS 3919
NC 98-7-1
ORUS 3916
ORUS 3918
ORUS 3883
ORUS 3906
ORUS 3910
ORUS 3911
ORUS 3912
ORUS 3884
ORUS 3898
ORUS 3902
ORUS 3926
ORUS 3930
ORUS 3931
ORUS 3929
ORUS 3777
ORUS 3778
‘Allen’
‘Black Hawk’
‘Black Knight’
‘Bristol’
‘Cumberland’
‘Dundee’
‘Earlysweet’
553773
‘Ebonee’
Origin
Davidson County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
DeKalb County, TN
Grundy County, TN
Grundy County, TN
Henderson County, TN
Roane County, TN
Unicoi County, TN
Unicoi County, TN
Van Buren County, TN
Van Buren County, TN
Van Buren County, TN
Van Buren County, TN
Van Buren County, TN
Warren County, TN
Warren County, TN
Warren County, TN
Columbia County, WI
Inwood, WV
Preston County, WV
Shepherdstown, WV
Mactaquac, NB, Canada
Simcoe, ON, Canada
‘Bristol’ × ‘Cumberland’, 1957
‘Quillan’ × ‘Black Pearl’, 1955
‘Johnson Everbearing selfed’, 1973
‘Watson Prolific’ × ‘Honeysweet’, 1934
Wild selection from Pennsylvania, 1890s
‘Smith1’ × ‘Palmer’, 1927
(‘Haut’ × R. leucodermis) × open-pollinated,
1996
‘Cumberland’ × open-pollinated, 1961
Type
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Wild
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
121
Table 5.1. (continued)
z
PI no.
658341
Name
‘Explorer’
553768
553769
553742
553736
553772
618387
553740
553741
553737
553738
618505
618458
‘Hanover’
‘Haut’
‘Jewel’
‘Huron’
‘John Robertson’
‘Mac Black’
‘Munger’
‘New Logan’
‘Plum Farmer’
‘Shuttleworth’
‘Somo’
‘White Chimera’
Origin
Wild parents from New York and Arkansas,
2004
Unknown, perhaps from Indiana
‘Manteo’ selfed × ‘Bristol’ selfed, 1987
(‘Bristol’ × ‘Dundee’) × ‘Dundee’, 1973
‘Rachel’ × ‘Dundee’, 1965
Wild selection from near Hot Springs, SD, 1934
Unknown
Reputed to be ‘Schaefer’ open-pollinated
Unknown wild parentage
Chance seedling from Ohio, 1892
Developed in New York, 1933
Unknown, from wild parents, 1956
Sport of a ‘Munger’ seedling, 1993
Type
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
Cultivar
- Accessions not yet available through the USDA, ARS, National Genetic
Resources Program. Germplasm Resources Information Network (GRIN).
Table 5.2. Summary information for 21 SSR primer pairs used for studying genetic diversity in wild and cultivated black
raspberry (Rubus occidentalis L.) genotypes. Names of primers evaluated with the M13 sequence at the start of the forward
primer are italicized.
Primer name
ssrRhCBA23zy
Rubus 110ayx
RhM003zy
Rub1C6zy
Rh_ME0013bG01zw
RubFruitC1yw
Rubus 275ayx
Rubus 270ayx
RO_CBEa010N20 w
Rh_ME0013cE02zw
Rubus 262byx
Rubus 123ayx
Rh_ME0015cH02zw
Rh_ME0013cF08zw
Allele size range in
R. occidentalis (bp)
98 – 154
Allele size range in
R. leucodermis (bp)
110 – 114
163 – 215
161
Graham et al. 2004
(TG)10
211 – 229
194 – 198
Castillo et al. 2010
(CT)15
237 – 268
235
Dossett et al. 2010
(GA)38
243 – 251
255 – 259
Lewers et al. 2008
CTT-(CCT)7
161 – 164
158
Graham et al. 2004
(AG)27
112 – 162
156 – 170
Graham et al. 2004
(GA)10
153 – 171
159 – 161
Graham et al. 2004
(GA)9
114 – 118
114 – 118
Unpublished
(TA)8
318 – 326
325
Lewers et al. 2008
(AG)15
203 – 209
203
Graham et al. 2004
(AG)8
158 – 188
162
Graham et al. 2004
(TC)9
212 – 216
214 – 216
Lewers et al. 2008
(TC)15
248 – 278
252 – 256
Lewers et al. 2008
(AG)14
233 – 247
231
Motif
(GA)10G(GA)5
(TC)8
Source
Lopes et al. 2006
Unpublished
122
RO_CBEa011M1w
Primer sequence
F: ATCGGGGATTTGGTGTGGGTTTAGG
R: ATTGTGTGCATCACTCTGAGAACCG
F: AAACAAAGGATAAAGTGGGAAGG
R: TGTCAGTTGGAGGGAGAACA
F: CCATCTCCAATTCAGTTCTTCC
R: AGCAGAATCGGTTCTTACAAGC
F: TCTGCCTCTGCATTTTACACAG
R: GTTTAGGTAAGCAATGGGAAAGCTC
F: CCCTCCATCTCCACCATAAA
R: GTAAGGCCACCCCATTGAG
F: CACGAGCTTCATCCTCTTCC
R: ATCCAAAGCTTTTGCGATTG
F: CACAACCAGTCCCGAGAAAT
R: CATTTCATCCAAATGCAACC
F: GCATCAGCCATTGAATTTCC
R: CCCACCTCCATTACCAACTC
F: GGGGGCTTTACATCATCATT
R: TTCGTAGTCTTGCCCTTGCT
F: AGGGTGGGTCTGAGATTGTG
R: AACAGTGCACAGGGGCTAAT
F: TGCATGAAGGCGATATAAAGG
R: TCCGCAAGGGTTGTATCCTA
F: CAGCAGCTAGCATTTTACTGGA
R: GCACTCTCCACCCATTTCAT
F: TGGATTTCCACACGCACATA
R: TGTTGGATTTGCCTCCTTTC
F: TTTGTCTCCGTCTTTTTGCC
R: CCTCCGAAGAAAAACAGCAG
F: TCGAACCTGTTGCCTTCTCT
R: TCCATTTCCAAAACACATTGA
Table 5.2. (continued)
Primer name
Rh_ME0007aB01zw
Rubus 223ayx
Rubus 26ayx
Rubus 126byx
Rubus 107ayx
Rubus 194h yx
Primer sequence
F: TGGTGGTTCACCGTTCACTA
R: GAAATGCTTGAAGCCGAGAG
F: TCTCTTGCATGTTGAGATTCTATT
R: TTAAGGCGTCGTGGATAAGG
F: AACACCGGCTTCTAAGGTCT
R: GATCCTGGAAAGCGATGAAA
F: CCTGCATTTTTCTGTATTTTGG
R: TCAGTTTTCTTCCCACGGTTA
F: GCCAGCACCAAAAACCTACA
R: TTTCACCGTCAAGAAGAAAGC
F: TGTGTTGTTCTCTGCAACCA
R: AGCCCTTACTTTTCCTGCAA
Allele size range in
R. occidentalis (bp)
145 – 155
Allele size range in
R. leucodermis (bp)
147 – 149
Source
Lewers et al. 2008
(AT)4-(TA)8(AT)10
(CT)11-(CA)29
156 – 166
160 – 162
Graham et al. 2004
123 – 143
129 – 133
Graham et al. 2004
(CT)31-(CA)22
152 – 174
180 – 182
Graham et al. 2004
(AG)8
160 – 164
160
Graham et al. 2004
(GA)12
127 – 133
131 – 133
Graham et al. 2004
Motif
(CT)15
z
- SSR marker developed in blackberry.
- SSR marker developed from a genomic library.
x
- SSR marker developed in red raspberry.
w
- SSR marker developed from an expressed sequence tag (EST) library.
y
123
Table 5.3. Allelic diversity, expected heterozygosity (He), observed heterozygosity (Ho) and polymorphism information
content (PIC) for 21 Rubus SSR primer pairs in 21 cultivars and 125 wild black raspberry (R. occidentalis L.) accessions.
Cultivars (n=21)
Primer name
ssrRhCBA23
Wild accessions (n=125)
All genotypes (n=146)
Allele #
3
He
0.54
Ho
0.67
PIC
0.44
Allele #
23
He
0.91
Ho
0.35
PIC
0.90
Allele #
24
He
0.90
Ho
0.40
PIC
0.89
Rubus 110a
4
0.68
0.76
0.63
22
0.88
0.32
0.87
22
0.88
0.38
0.87
RhM003
3
0.56
0.81
0.49
6
0.47
0.22
0.42
6
0.52
0.31
0.45
Rub1C6
4
0.57
0.71
0.50
18
0.90
0.41
0.90
18
0.89
0.45
0.88
Rh_ME0013bG01
2
0.13
0.14
0.12
3
0.23
0.08
0.21
3
0.22
0.09
0.20
RubFruitC1
2
0.24
0.29
0.21
2
0.23
0.10
0.20
2
0.23
0.13
0.20
Rubus 275a
5
0.66
0.76
0.60
20
0.91
0.35
0.90
20
0.90
0.41
0.89
Rubus 270a
2
0.44
0.57
0.35
9
0.78
0.23
0.74
9
0.77
0.28
0.73
RO_CBEa010N20
2
0.17
0.19
0.16
2
0.39
0.16
0.31
2
0.36
0.16
0.30
Rh_ME0013cE02
3
0.50
0.67
0.40
5
0.56
0.17
0.46
5
0.55
0.24
0.45
Rubus 262b
2
0.09
0.10
0.09
1
0.00
0.00
0.00
2
0.01
0.01
0.01
Rubus 123a
2
0.09
0.10
0.09
4
0.51
0.21
0.41
5
0.49
0.19
0.39
Rh_ME0015cH02
1
0.00
0.00
0.00
3
0.10
0.07
0.09
3
0.09
0.06
0.08
Rh_ME0013cF08
4
0.48
0.52
0.43
16
0.81
0.32
0.79
16
0.78
0.35
0.76
RO_CBEa011M11
3
0.48
0.57
0.38
5
0.61
0.19
0.54
5
0.61
0.25
0.55
Rh_ME0007aB01
4
0.54
0.62
0.44
6
0.57
0.27
0.52
6
0.57
0.32
0.52
Rubus 223a
1
0.00
0.00
0.00
6
0.54
0.25
0.51
6
0.48
0.21
0.46
Rubus 26a
4
0.64
0.86
0.57
9
0.71
0.22
0.66
9
0.70
0.32
0.65
124
Table 5.3. (continued)
Cultivars (n=21)
Primer name
Rubus 126b
Wild accessions (n=125)
All genotypes (n=146)
Allele #
3
He
0.56
Ho
0.67
PIC
0.49
Allele #
10
He
0.66
Ho
0.30
PIC
0.62
Allele #
10
He
0.66
Ho
0.36
PIC
0.61
Rubus 107a
1
0.00
0.00
0.00
3
0.10
0.04
0.10
3
0.09
0.03
0.08
Rubus 194h
2
0.17
0.19
0.16
5
0.46
0.14
0.38
5
0.43
0.15
0.36
2.7
0.36
0.44***
0.31
8.5
0.54
0.21***
0.5
8.6
0.53
0.24***
0.49
Mean:
***
Significant at P < 0.0001.
125
Table 5.4. Microsatellite alleles (fragment size in bp) at six loci in ‘Jewel’, ‘Haut’, ‘Allen’, ‘Earlysweet’, ‘Black Knight’,
‘Mac Black’ and related black raspberry (Rubus occidentalis L.) cultivars, illustrating shared rare alleles and discrepancies in
reported pedigrees. Names of cultivars with pedigree discrepancies are in bold and are presented in a block with their
reported parents and clones sharing unique alleles. Alleles specifically mentioned in the text are in bold and underlined.
Fingerprints for some cultivars (i.e. Dundee, Huron and Haut) are repeated in different blocks for ease of comparison.
Cultivar
Bristol
Dundee
Huron
Jewel
Bristol
Cumberland
Huron
Haut
Allen
Ebonee
Haut
Dundee
Earlysweet
Black Knight
Mac Black
ssrRhcBA23
124, 126
124
112, 124
112, 124
124, 126
124, 126
112, 124
124
124
124, 126
124
124
124, 126
126
124, 126
Rubus 275a
116, 144
116, 132
128, 144
144
116, 144
116, 144
128, 144
128, 132
144
116
128, 132
116, 132
116, 144
134, 144
144
Rubus 262b
203
203
203
203
203
203
203
203
203
203
203
203
203
203, 209
203, 209
Rubus 123a
158
158, 188
158
158
158
158
158
158
158
158
158
158, 188
158, 188
158
158
Rubus 126b
154, 168
158, 168
168
158, 168
154, 168
154, 168
168
168
158
154, 168
168
158, 168
154, 168
154, 168
168
Rubus 110a
183, 185
183
169, 183
169, 183
183, 185
183, 185
169, 183
187
183, 185
187
187
183
183, 187
169, 183
169, 183
126
127
Fig. 5.1. Geographical distribution of 137 wild black raspberry (Rubus occidentalis
L.) populations surveyed.
is
erm
od
E
uc
,N
s le
ark , NC
P
bu
n IN
te
Ru
Sta ebulo o., IL
Z m C o.,
ron
-3
ad
a
Ch 4-10 utn an C
P eg
8
38
41
NC 794 auk
3
W
US
2
OR 1.00
64
UB
CR
Cultivars
US
OR
ORUS
4134
Pibel
ORU
Lake
S
State
ORU 3811 Alle
Recre
S
gany
ation A
Co.,
ORU 3833 C
rea, N
MD
assvi
E
'Ex S 3830
lle, M
plore
F
O
o
rdla
OR
r'
n
U
d, M
OR S 385
O
OR US 37 6 Ho
wa
rd,
OR US 4 98 S
PA
ulliv
OR US 3 112
a
OR US 926 Okem n Co.,
IN
os
Co
OR US 393
,
US 379 0 In lumb MI
ia
41 9 F woo
Co
24
d,
ar
.
,
WI
Bo mer WV
s
nn
er burg
,
Sp
rin IN
gs
,K
S
128
SD
rk,
Pa
A
e
,M
ov gton
Gr
in
Y
ion Barr ls, N
n
l
U
at
Fa
43 Gre ake
1
4
4
I
op
US 380 3 C th, M
., IL
PA
OR US 384
Co
Ba
gs,
OR US 109 Sprin kegan
au
OR US 4 853
N
W
T
o.,
OR US 3 2.001
eC
OR B 64 Roan
NY
U
va,
CR 98-7-1
ene
G
AL
ne,
to
NC S 3840
n
e
PA
U
3M
Co.,
OR
ir, NJ
412
ene
S
re
servo
U
G
n Re
OR
52
ua
8
3
asq
S
an
M
A
ORU
5
P
S 395
brook,
ORU
Honey
3851
ge, PA
e Colle
ORUS
MI
54 Stat
38
S
te Forest,
ORU
Russ Sta
48 Fred
ORUS 39
idson Co., TN
Dav
3
386
ORUS
Falls, OH
ORUS 4108 Newton
ate
St
0.1
ORUS 3902 Smithville, TN
ORUS 3832 Fordland, MO
ORUS 4113 Glas
sy Mountain, SC
ORUS 4137
Valentine, NE
ORUS 37
80 Rolan
d, IA
ORUS
4107 H
illiard, O
ORUS
H
4139
Chad
ORU
ron, N
S
E
ORU 4150 Ha
S
sty, M
ORU 3819 H
N
S3
allow
915
OR
ell, M
US
C
E
he
380
OR
U
0 E atham
Co.,
OR S 38
llisto
4
TN
n, IN
OR US 41 1 Ge
n
U
2
eva
OR S 4 0 C
, NY
lay
OR US 117
to
OR US 4119 Clay n, GA
Cla ton,
OR US 380
US 38 6 H yton GA
ow
,G
41 05
A
40 Wa ard
Pe shin Co.
as
gto , MD
eC
nC
re
ek o., M
St
ate D
Pa
rk
,S
D
OR
OR
US
US
OR
38
39
US
06
78
OR
38
83 Van Smit
US
Va Bur hvi
39
n
1
ll
HD
e
F-0 1 Va Bur n Co e, TN
39
n B en
OR
C ., T
Ap
US
pala uren o., T N
C
3
N
869
ch
OR
o.
US
De ian tr , TN
389
ORU
8 W kalb C ail, G
A
S3
o.,
arre
871
T
n
Co., N
Dek
ORU
a
T
S 41
49 B lb Co., N
ORU
TN
elgra
S 38
de,
7
3
Smit
M
ORU
hville, N
S 391
6 Uni
TN
coi C
ORUS
o., TN
3904
Woodb
ury, TN
ORUS
3919 As
heville,
ORUS 38
TN
38 Rutherfo
rd Co., NC
ORUS 3802 Faye
tte Co., KY
ORUS 3812 Ann Arundel Co.,
MD
ORUS 3951 Poughkeepsie, NY
ORUS 3918 Unicoi Co., TN
herford Co., NC
ORUS 3837 Rut
C
., N , SD
Co
lls
on x Fa
SC
u
dis
in,
Ma st Sio unta
o
a
35
38 5 E ich M , ON
US 414 5 R
coe ti, OH
im
a
OR S 11
U
S
n
B
4
cin
,N
C
OR US 778
c
S
Cin
in,
qua
a
OR US 3 849
unta
act
Mo
WV
OR US 3 77 M
I
ssy
wn,
37
st, M
OR
Gla
rdsto
US
Fore
14
phe
tate
OR S 41
She
U
ss S
OR S 3929 red Ru
F
MD
ORU 3949
ood,
S
inkw
ORU 3809 L
n, NY
S
ertow
ill
ORU
42 M
ills, SD
H
S 38
ewton
ORU
N
4144
Co., TN
ORUS
vidson
Refuge, MN
3864 Da
al Wildlife
ORUS
ne Nation
8 Big Sto
ORUS 414
OR
US
OR 394
US 6 W
37
a
81 uke
O
OR
US RUS Iriq gan
41
41 uois Co
OR
2
2
5
US
6
C .,
P
Alm o., IL
41
OR
I
21 erry
US
La a, K L
Un
OR
3
k
8
io
US
26
n C e, K S
381
Ro
o., S
s
0
e
G
OR
How
vil
A
US
ard le, M
379
ORU
N
Co
6B
., M
S 38
loo
D
67
ORU
Dow mfield,
S 38
e
IN
15 M lltown,
ORU
T
onm
S 41
outh N
85 C
, ME
harels
ORUS
town
3839
, RI
Gene
ORUS
va, NY
3821 Ca
mden, M
ORUS 38
E
07 Washi
ngton Co.,
MD
ORUS 3820
East Vassalb
oro, ME
ORUS 3814 Orono, ME
ORUS 3912 Van Buren Co., TN
NC 98-12-1 Dekalb Co., TN
Riley, KS
ORUS 4129 Fort
, KS
Manhattan
ORUS 4127
rk, SD
State Pa
D
d Lakes
oo
le
kw
Va , M
46 Oa
3808
ORUS 41
ORUS
y, NE
Halse
36
N
M
41
ount,
ORUS
osem er, ME
28 R
ardin NE
S 38
G
U
R
O
17
ity,
S 38
aC
NE
ORU
rask rings, E
Neb
p
N
ia S oup,
.001
r
1
3
to
L
IN
ic
B 17
rth
o.,
5V
No
CRU
s C n, KS
413
3
k
3
S
ic de
U
41
MI
ndr
OR
US
He 8 Og ove, N
OR
7
,T
12
79
Gr
S3
S 4 Oak pring ., TN
U
U
OR
OR
47 tt's S n Co . , TN
39
u
e Co
r
c
S
r
U
a
rth
y
OR
No 4 W und
89 388
Gr
38
S
93
S
U
8
U
OR S 3
OR
U
OR
Fig. 5.2. Unweighted pair-group method analysis (UPGMA) dendrogram depicting
all black raspberry (Rubus occidentalis L.) genotypes studied. A cluster containing
most black raspberry cultivars and a few wild accessions has been collapsed and is
depicted in Fig. 5.3.
129
70
88
77
ORUS 3931 Preston Co., WV
ORUS 3844 Dundee, NY
ORUS 3857 Davidson Co., TN
ORUS 3824 St. Paul, MN
ORUS 3816 West Kennebunk, ME
‘Dundee’
‘Allen’
ORUS 3956 Tom's River, NJ
98
‘Jewel’
‘John Robertson’
‘Hanover’
‘Ebonee’
‘Earlysweet’
‘Mac Black’
‘Black Knight’
‘Huron’
ORUS 4110 Benton Harbor, MI
ORUS 4130 Minneapolis, KS
‘Black Hawk’
ORUS 3801 southern IN
66
‘Haut’
‘Somo’
‘White Chimera’
‘Cumberland’
‘Bristol’
‘New Logan’
‘Munger’
‘Shuttleworth’
‘Plum Farmer’
0.05
Fig. 5.3. Unrooted neighbor-joining dendrogram of black raspberry (Rubus
occidentalis L.) cultivars and closely paired wild accessions from a condensed
cluster in Fig. 5.2. Numbers near nodes show bootstrap support for pairings (percent
of 1,000 trees).
130
Fig. 5.4. Principal components plot of wild and cultivated black raspberry (Rubus
occidentalis L.) based on Euclidean distance measured from 21 polymorphic SSR
loci and illustrating PCA clustering of wild accessions, black raspberry cultivars,
wild accessions clustered with cultivars in Fig. 5.3, and wild accessions clustering
with ‘Explorer’ in Fig. 5.2.
131
Chapter 6:
Conclusions
Michael Dossett
132
Over the last 180 years since the domestication of black raspberry, breeders
have made impressive progress in selecting for and breeding vigorous plants with
good fruit size from wild germplasm. The cultivars examined in Chapter 3 were
clearly superior to most of the wild seedlings in a variety of horticultural traits,
particularly fruit weight. Despite this, these cultivars are only a few generations
removed from wild plants and illustrate what may be possible from careful breeding
of superior wild selections. The results of the preceding chapters have clearly
demonstrated that there is a tremendous amount of genetic diversity present in wild R.
occidentalis germplasm that is not represented by black raspberry cultivars. More
importantly, the insect and disease resistance found in this germplasm is in stark
contrast to the material available to breeders until now.
The aphid resistance described in Chapter 2 promises to be a major step
toward reducing the virus problems currently experienced by the black raspberry
industry. Managing the deployment of these resistance genes in new cultivars will be
critical to maintaining their durability in the future. The development of molecular
tools for identifying and selecting for these resistance genes would be tremendously
valuable for this purpose, and would significantly aid in preventing the breakdown of
resistance that has occurred in red raspberry.
Verticillium wilt is also a major problem for the industry. This disease has
been underrecognized and misdiagnosed for many years. New germplasm sources,
particularly from the southeastern part of the range, seemed to have good tolerance
in the field, but a lot of questions remain. Whether this germplasm represents actual
133
resistance to the pathogen or whether the increased vigor of these selections simply
makes them less susceptible to disease progression is still not known. Inheritance of
resistance to this pathogen is also not well understood. There is good evidence in the
literature for major gene control (Wilhelm and Thomas, 1950; Wilhelm et al., 1965)
as well as for multigenic resistance (Fiola and Swartz, 1994). Crosses with red
raspberry seem to transmit resistance to infection, however backcrossing has not
been very successful in transferring this trait to black raspberry (Keep, 1989). A
reliable method of screening black raspberry germplasm for resistance to
Verticillium wilt needs to be developed to address these questions. A screening
procedure must be simple, inexpensive, and relatively fast in order to be of utility to
the breeding program. The nature of the disease and the rate of turnover in seedling
fields being evaluated mean that breeding for resistance to Verticillium wilt will
likely remain a challenge until a reliable and economically feasible screening method
is developed.
Because of the demands of the processing industry and the interest in black
raspberry for nutraceutical product development, anthocyanin content and
composition are among the most critical indicators of processed black raspberry fruit
quality. Despite this, relatively little is known about the genetic control and
regulation of anthocyanin production in black raspberry fruit. The identification of a
pigment mutant in black raspberry provides a valuable opportunity to map, identify,
and study one of these genes and its affect not only on the anthocyanin composition
but on total anthocyanin content as well. Ongoing analysis of black raspberry
134
anthocyanin content and other fruit chemistry traits promises to shed more light on
genetic diversity available for selection and breeding.
A molecular analysis of genetic diversity in wild black raspberry germplasm
supports the phenotypic diversity observed in these populations. Black raspberry
cultivars appear to be more closely related to each other than to most of the wild
populations examined. At the same time, this analysis has revealed a number of
discrepancies between the microsatellite fingerprints of several cultivars and their
reported pedigrees. Some observations in the field were also indicative of
mislabeling of plants in the industry and this is an area needing further study. At the
very least, consistency in labeling and identification is important to the breeding
program not only for deciding which parents to use in crosses but also for the
identification of standards used to judge the performance of advanced breeding
selections. The application of molecular tools in black raspberry will be beneficial
not only for the reliable identification of plants, but also for studying diversity in the
germplasm to identify the best strategies for future collection and germplasm
preservation efforts.
Despite the apparent diversity in wild black raspberry germplasm and its
potential for the development of superior, disease resistant cultivars, significant
challenges for the black raspberry industry remain. Expansion of the industry will be
critical to the stabilization of prices and the long-term viability of the industry. New
cultivars with superior disease resistance and durability will be the first step in giving
new growers confidence in the profitability of growing black raspberry. At the same
135
time, cultivars with greater longevity will reduce the turnover in fields and have a
stabilizing effect on acreage and prices in the market.
Adopting good clean-plant practices will also be critical for growers to be
successful. At present, it is a common practice for black raspberry growers to
propagate their own planting stock from one-year-old fields by letting cane tips root
in the fall, and then digging them in the spring to move to new plantings. It is easy to
understand why growers take this approach to reduce the cost of establishing new
fields when a new planting from clean stock will begin to decline in a year or two
anyway. However, the result is that growers spread viruses and soil-borne pathogens
from one field to the next before a field is even established.
In his classic “The Small Fruits of New York”, Hedrick (1925) may well
have been right when he wrote “The future of the black raspberry is a most
promising one.” While nearly 90 years have passed since Hedrick’s assessment,
evaluation of wild germplasm shows tremendous potential for this crop. During the
course of this work more than 60 selections have been made for further evaluation
and crossing. Many of these have excellent vigor and/or disease resistance, some are
very early or very late ripening, and others still have exceptional fruit firmness, semismooth canes, or other novel qualities. These selections will advance breeding efforts
and help ensure the continued viability and sustainability of the black raspberry
industry.
136
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